School of Science and Technology, Georgia Gwinnett College, 1000 University Center Lane, Lawrenceville, GA 30043, USA.
Biomolecules. 2019 May 8;9(5):175. doi: 10.3390/biom9050175.
Neuromelanin, the polymeric form of dopamine which accumulates in aging neuronal tissue, is increasingly recognized as a functional and critical component of a healthy and active adult human brain. Notorious in plant and insect literature for their ability to bind and retain amines for long periods of time, catecholamine polymers known colloquially as 'melanins' are nevertheless curiously absent from most textbooks regarding biochemistry, neuroscience, and evolution. Recent research has brought attention to the brain pigment due to its possible role in neurodegeneration. This linkage is best illustrated by Parkinson's disease, which is characterized by the loss of pigmented dopaminergic neurons and the 'white brain' pathological state. As such, the ability to determine the binding affinity of neurotoxic agents, as well as any potential specific endogenous ligands to neuromelanin are of interest and potential value. Neuromelanin has been shown to have saturable binding interactions with nicotine as monitored by a fluorimeter. This interaction provides a signal to allow for a competition-binding assay with target molecules which do not themselves produce signal. The current report establishes the viability of this competition assay toward three compounds with central relevance to Parkinson's disease. The K of binding toward neuromelanin by methyl-phenyl-pyridinium ion (MPP+), dopamine, and 6-hydroxydopamine were found to be 1 mM, 0.05 mM, and 0.1 mM, respectively in the current study. In addition, we demonstrate that 6-hydroxydopamine polymerizes to form neuromelanin granules in cultured dopaminergic neurons that treated with 2,4,5-trihydroxy-l-phenylalanine. Immunohistochemical analysis using fluor-tagged anti-dopamine antibodies suggests that the incorporation of 6-hydroxydopamine (following internalization and decarboxylation analogous to levodopa and dopamine) alters the localized distribution of bound dopamine in these cells.
神经黑色素是多巴胺的聚合形式，在衰老的神经元组织中积累，它越来越被认为是健康活跃的成年人脑的功能和关键组成部分。在植物和昆虫文献中，儿茶酚胺聚合物因其能够长时间结合和保留胺而臭名昭著，俗称“黑色素”，但在大多数关于生物化学、神经科学和进化的教科书中却奇怪地缺失。最近的研究引起了人们对脑色素的关注，因为它可能在神经退行性变中起作用。帕金森病就是最好的例证，这种疾病的特征是色素性多巴胺能神经元丧失和“白脑”病理状态。因此，确定神经毒性剂的结合亲和力以及任何潜在的特定内源性配体与神经黑色素的结合能力是很有意义和潜在价值的。荧光光度计监测表明，神经黑色素与尼古丁具有饱和结合相互作用。这种相互作用提供了一个信号，允许与目标分子进行竞争结合测定，而目标分子本身不产生信号。本报告确立了这种竞争测定法对三种与帕金森病密切相关的化合物的可行性。在本研究中，发现甲基-苯-吡啶鎓离子（MPP+）、多巴胺和 6-羟多巴胺与神经黑色素的结合 K 值分别为 1mM、0.05mM 和 0.1mM。此外，我们还证明 6-羟多巴胺在 2,4,5-三羟基-l-苯丙氨酸处理的培养多巴胺能神经元中聚合形成神经黑色素颗粒。用荧光标记的抗多巴胺抗体进行免疫组织化学分析表明，6-羟多巴胺（类似于左旋多巴和多巴胺的内化和脱羧）的掺入改变了这些细胞中结合多巴胺的局部分布。
