Blood Genomics and Cell Model Approaches for Neuropsychiatric Disorders

今年，我们的团队与GCAP转基因实验室合作，研究儿茶酚-O-甲基转移酶（COMT）的分布和定位。COMT是精神分裂症的危险基因，其酶产物对多巴胺和其他儿茶酚化合物的失活和代谢至关重要。COMT产生2种转录本，其中长型转录本被翻译成膜结合型（MB）和可溶型（S）。 MB-COMT主要存在于大脑中，并与多巴胺信号传导有关，而S-COMT存在于外周组织中，并在肝脏和血液中的解毒和儿茶酚降解中发挥作用。目前，没有MB-COMT特异性抑制剂。托卡朋是一种与L-多巴胺一起用于治疗帕金森病的药物，用于研究多巴胺依赖性脑功能，抑制MB-COMT和S-COMT。S-COMT的抑制可能在肝毒性中起作用，这限制了其治疗用途。因此，开发MB-COMT特异性抑制剂将是有利的。COMT基因Val 158 Met多态性是影响COMT活性的重要基因。met等位基因与较低的酶活性和较好的认知功能有关，但增加了对疼痛的敏感性。在COMT中存在影响酶活性的其他功能多态性的知识使得基于儿茶酚的药物的个体化剂量相当合理。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表征了COMT在皮质介导的认知中的作用。托卡朋的临床评估表明皮质功能的改善。然而，鉴于所有关于COMT的信息，MB-COMT的细胞生物学仍然不清楚。本研究检测了MB-COMT在人淋巴母细胞样细胞中的取向和MB-COMT在锥体神经元上的细胞分布。为了确定MB-COMT的方向，我们使用了转染的人淋巴母细胞，这导致MB-COMT在两端用绿色荧光蛋白（GFP）标记。我们的结果显示在用与R-藻红蛋白缀合的抗GFP抗体处理后仅C-末端GFP标签。这一发现表明MB-COMT的C-末端在细胞外，并且C-末端区域中的催化结构域在细胞外空间中。这种取向使得MB-COMT能够催化甲基从S-腺苷甲硫氨酸转移到突触和细胞外空间中的儿茶酚胺，同时不影响跨膜转运。 这表明新开发的对细胞膜不可渗透的COMT抑制剂在脑中将是MB-COMT特异性的。未来的研究将进行，这将有助于开发新的治疗化合物，用于治疗复杂的精神疾病。

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.