Blood Genomics and Cell Model Approaches for Neuropsychiatric Disorders

今年，我们小组与GCAP转基因实验室合作，研究了儿茶酚- o -甲基转移酶（COMT）的分布和取向。COMT是一种精神分裂症风险基因，其酶产物对多巴胺和其他儿茶酚类化合物的失活和代谢至关重要。COMT产生两种转录本，其中长形式被翻译成膜结合形式（MB）和可溶性形式(S)。MB-COMT主要存在于大脑中，与多巴胺信号传导有关，而S-COMT存在于外周组织中，在肝脏和血液中的解毒和儿茶酚降解中起作用。目前，还没有MB-COMT特异性抑制剂。Tolcapone是一种用于治疗帕金森病的l -多巴胺药物，用于研究多巴胺依赖的脑功能，抑制MB-COMT和S-COMT。S-COMT的抑制可能在肝毒性中起作用，这限制了其治疗应用。因此，开发MB-COMT特异性抑制剂将是有利的。COMT基因具有很好的功能多态性Val158Met，影响人类COMT活性。met等位基因与较低的酶活性和较好的认知功能有关，但会增加对疼痛的敏感性。在COMT中存在影响酶活性的其他功能多态性的知识，使得个体化剂量的儿茶酚类药物非常合理。临床脑疾病分支/基因、认知和精神病项目以及其他实验室已经很好地描述了COMT在皮质介导的认知中的作用。临床评估表明，托尔卡彭可以改善皮质功能。然而，鉴于所有关于COMT的信息，MB-COMT的细胞生物学仍不清楚。本研究检测了MB-COMT在人淋巴母细胞样细胞中的定向和MB-COMT在锥体神经元上的细胞分布。为了确定MB-COMT的定向，我们使用转染的人淋巴母细胞样细胞，结果在MB-COMT的两端都标记有绿色荧光蛋白（GFP）。我们的结果显示，在与r -藻红蛋白偶联的抗GFP抗体处理后，只有c端GFP标签。这一发现表明MB-COMT的c端位于细胞外，c端区域的催化结构域位于细胞外空间。这种取向使得MB-COMT能够在突触和细胞外空间催化甲基从s-腺苷蛋氨酸转移到儿茶酚胺，而不影响跨膜运输。这表明新开发的不渗透细胞膜的COMT抑制剂在大脑中是MB-COMT特异性的。未来的研究将有助于开发治疗复杂精神疾病的新治疗化合物。

This year, our group collaborated with the GCAP transgenic lab to study the distribution and orientation of catechol-O-methyltransferase (COMT). COMT is a schizophrenia risk gene and its enzyme product is critical for the inactivation and metabolism of dopamine and other catechol compounds. COMT produces 2 transcripts with the long form is translated into the membrane-bound (MB) and the soluble (S) form. MB-COMT is primarily found in the brain and is implicated in dopamine signaling, whereas S-COMT is found in peripheral tissues and has a role in detoxification and catechol degradation in the liver and blood. Currently, there is no MB-COMT specific inhibitor. Tolcapone, a drug with L-Dopamine for treatment of Parkinsons disease, is used to study dopamine-dependent brain functions inhibits both MB-COMT and S-COMT. The inhibition of S-COMT may play a role in liver toxicity which limits its therapeutic use. Therefore, it will be advantageous to develop MB-COMT specific inhibitors. COMT gene has a well characterized functional polymorphism, Val158Met, which effects COMT activity in humans. The met allele is associated with having much lower enzyme activity and better cognitive function but increases sensitivity to pain. The knowledge that there exist other functional polymorphisms in COMT that effect enzyme activity, makes individualized doses of catechol-based drugs quite reasonable. The Clinical Brain Disorders Branch/Genes, Cognition and Psychosis Program as well as other labs have well characterized the role of COMT in cortically mediated cognition. Clinical assessments of tolcapone have suggested improvement of cortical function. However, given all the information on COMT, the cell biology of MB-COMT remains unclear. This study examined MB-COMT orientation in human lymphoblastoid cells and cellular distribution of MB-COMT on pyramidal neurons. To determine the orientation of MB-COMT, we used transfected human lymphoblastoid cells, which resulted in MB-COMT tagged on either terminus with green fluorescent protein (GFP). Our results showed only the C-terminal GFP tag upon treatment with anti-GFP antibody conjugated with R-Phycoerythrin. This finding indicated that the C-terminus of MB-COMT is outside of the cell and that the catalytic domain in the C-terminal region is in the extracellular space. This orientation renders MB-COMT capable of catalyzing the transfer of a methyl group from S-adenosylmethionine to catecholamines in synaptic and extracellular spaces, while not impacting cross-membrane transport. This suggests that newly developed COMT inhibitors impermeable to the cell membrane will be MB-COMT specific in the brain. Future studies will be conducted which will aid in the development of new therapeutic compounds for treatment of complex psychiatric disorders.