Development of a Competition-Binding Assay to Determine Binding Affinity of Molecules to Neuromelanin via Fluorescence Spectroscopy

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_d 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.