Abstract
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 Kd 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.
Highlights
We have found that synthetic Neuromelanin and Parkinson’s DiseaseNeuromelanin (NM) ‘spiked’ with 5% of the normal dopamine replaced by 6-hydroxydopamine has unique properties compared to ‘normal’ NM, inasmuch as it exhibits a much higher propensity to form a hydrogel state
Our longstanding interest in the measurement of the binding affinity of dopamine to synthetic NM presents additional problems in that the NM polymer is derived from that very dopamine monomer, such that the dopamine being tested can conceivably add to the growing polymer rather than displaying equilibrium binding kinetics
The inhibitory constants of dopamine and 6-hydroxydopamine measured in the current study are at a level consistent with concentrations expected to be found in the cell
Summary
Neuromelanin (NM) is a pigment consisting of conjugated 5,6 dihydroxyindole rings resulting mainly from the spontaneous co-polymerization of dopamine and cysteine into a granule along with an apparent set of critical lipids and peptides that is normally found to accumulate in human brain tissue as a normal part of the aging process [1]. Adrenochrome spontaneously initiates the polymerization process in concentrated solutions and is the source of much of the oxidative stress in cells which harbor dopamine. A certain catecholamine threshold, the thiol antioxidant buffering defense mechanisms, consisting of glutathione and cysteine in redox equilibrium with their corresponding disulfide moieties, are overwhelmed, raising the oxidation state and allowing dopamine to proceed to the quinone. A spontaneous cascade of events is initiated which leads to polymer formation and the eventual appearance of neuromelanin granules [4,5]
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