Aminochrome Induces Disruption of Actin, Alpha-, and Beta-Tubulin Cytoskeleton Networks in Substantia-Nigra-Derived Cell Line

Abstract

In previous studies, we observed that cells treated with aminochrome obtained by oxidizing dopamine with oxidizing agents dramatically changed cell morphology, thus posing the question if such morphological changes were dependent on aminochrome or the oxidizing agents used to produce aminochrome. Therefore, to answer this question, we have now purified aminochrome on a CM-Sepharose 50-100 column and, using NMR studies, we have confirmed that the resulting aminochrome was pure and that it retained its structure. Fluorescence microscopy with calcein-AM and transmission electron microscopy showed that RCSN-3 cells presented an elongated shape that did not change when the cells were incubated with 50 muM aminochrome or 100 muM dicoumarol, an inhibitor of DT-diaphorase. However, the cell were reduced in size and the elongated shape become spherical when the cells where incubated with 50 muM aminochrome in the presence of 100 muM dicoumarol. Under these conditions, actin, alpha-, and beta-tubulin cytoskeleton filament networks became condensed around the cell membrane. Actin aggregates were also observed in cells processes that connected the cells in culture. These results suggest that aminochrome one-electron metabolism induces the disruption of the normal morphology of actin, alpha-, and beta-tubulin in the cytoskeleton, and that DT-diaphorase prevents these effects.

Molecular and Neurochemical Mechanisms Dopamine Oxidation To O-Quinones in Parkinson’s Disease Pathogenesis

“…Enzymatic GSH conjugation of dopamine o-quinone to 5-S-glutathionyl dopamine was catalyzed by GSTM2, although a non-enzymatic reaction has also been reported [43,55]. In contrast, other studies have involved aminochrome formation prior to the experiment [56] or the use of purified aminochrome [57][58][59][60][61][62]; thus, it is possible that 5,6-indolequinone had been formed in these experiments. However, the aminochrome conjugation with glutathione catalyzed by glutathione S-transferase M2 (GSTM2) generated only 4-S-glutathionyl-5,6-dihydroxyindoline and not 4-S-glutathionyl-5,6-dihydroxyindole, which should be the product of 5,6-indolequinone glutathione conjugation [63].…”

“…Additionally, it forms adducts with α-and β-tubulin, disrupting the cytoskeleton architecture and generating aggregates that completely change the cell shape and disrupt the cytoskeleton architecture further [58]. 5,6-Indolequinone also forms adducts with alpha synuclein [34].…”

“…This enzyme is expressed in astrocytes and dopaminergic neurons, and it is responsible for 97 % of the total quinone reductase activity in substantia nigra [83]. The two-electron reduction of aminochrome prevents aminochrome neurotoxic pathways because its inhibition or silencing with siRNA induces neurotoxicity both in cell cultures [56][57][58] or in vivo [81,82].…”

“…However, aminochrome has been reported to inactivate the proteasome [108,109]. Aminochrome forms adducts with α-and β-tubulin preventing the formation of microtubules required for autophagosome fusion with lysosomes and induces lysosome dysfunction by increasing its internal pH [58,59,110]. (iv) Aminochrome induces oxidative stress via hydroxyl radical formation (Fig.…”

The dopamine metabolite aminochrome inhibits mitochondrial complex I and modifies the expression of iron transporters DMT1 and FPN1

“…Aminochrome was prepared and used within 4 h by reacting dopamine with tyrosinase as described (Paris et al 2010). …”