Patricia Muñoz

Protective and toxic roles of dopamine in Parkinson's disease

The molecular mechanisms causing the loss of dopaminergic neurons containing neuromelanin in the substantia nigra and responsible for motor symptoms of Parkinsons disease are still unknown. The discovery of genes associated with Parkinsons disease (such as alpha synuclein (SNCA), E3 ubiquitin protein ligase (parkin), DJ‐1 (PARK7), ubiquitin carboxyl‐terminal hydrolase isozyme L1 (UCHL‐1), serine/threonine‐protein kinase (PINK‐1), leucine‐rich repeat kinase 2 (LRRK2), cation‐transporting ATPase 13A1 (ATP13A), etc.) contributed enormously to basic research towards understanding the role of these proteins in the sporadic form of the disease. However, it is generally accepted by the scientific community that mitochondria dysfunction, alpha synuclein aggregation, dysfunction of protein degradation, oxidative stress and neuroinflammation are involved in neurodegeneration. Dopamine oxidation seems to be a complex pathway in which dopamine o‐quinone, aminochrome and 5,6‐indolequinone are formed. However, both dopamine o‐quinone and 5,6‐indolequinone are so unstable that is difficult to study and separate their roles in the degenerative process occurring in Parkinsons disease. Dopamine oxidation to dopamine o‐quinone, aminochrome and 5,6‐indolequinone seems to play an important role in the neurodegenerative processes of Parkinsons disease as aminochrome induces: (i) mitochondria dysfunction, (ii) formation and stabilization of neurotoxic protofibrils of alpha synuclein, (iii) protein degradation dysfunction of both proteasomal and lysosomal systems and (iv) oxidative stress. The neurotoxic effects of aminochrome in dopaminergic neurons can be inhibited by: (i) preventing dopamine oxidation of the transporter that takes up dopamine into monoaminergic vesicles with low pH and dopamine oxidative deamination catalyzed by monoamino oxidase (ii) dopamine o‐quinone, aminochrome and 5,6‐indolequinone polymerization to neuromelanin and (iii) two‐electron reduction of aminochrome catalyzed by DT‐diaphorase. Furthermore, dopamine conversion to NM seems to have a dual role, protective and toxic, depending mostly on the cellular context.

Interactions of iron, dopamine and neuromelanin pathways in brain aging and Parkinson's disease.

HIGHLIGHTSIn this review the multiways interactions/modulations between iron, dopamine oxidation, neuromelanin and their role in brain are discussed.The relationship between iron and dopamine oxidation with consequences on cells is presented.An overview of synthesis and properties of peripheral melanins is given.Neuromelanin synthesis, structure and interaction with iron are reviewed, considering their protective/toxic pathways in neurons.The role of iron and neuromelanin and their possible detrimental effects in brain aging and Parkinsons disease are appraised. ABSTRACT There are several interrelated mechanisms involving iron, dopamine, and neuromelanin in neurons. Neuromelanin accumulates during aging and is the catecholamine‐derived pigment of the dopamine neurons of the substantia nigra and norepinephrine neurons of the locus coeruleus, the two neuronal populations most targeted in Parkinsons disease. Many cellular redox reactions rely on iron, however an altered distribution of reactive iron is cytotoxic. In fact, increased levels of iron in the brain of Parkinsons disease patients are present. Dopamine accumulation can induce neuronal death; however, excess dopamine can be removed by converting it into a stable compound like neuromelanin, and this process rescues the cell. Interestingly, the main iron compound in dopamine and norepinephrine neurons is the neuromelanin‐iron complex, since neuromelanin is an effective metal chelator. Neuromelanin serves to trap iron and provide neuronal protection from oxidative stress. This equilibrium between iron, dopamine, and neuromelanin is crucial for cell homeostasis and in some cellular circumstances can be disrupted. Indeed, when neuromelanin‐containing organelles accumulate high load of toxins and iron during aging a neurodegenerative process can be triggered. In addition, neuromelanin released by degenerating neurons activates microglia and the latter cause neurons death with further release of neuromelanin, then starting a self‐propelling mechanism of neuroinflammation and neurodegeneration. Considering the above issues, age‐related accumulation of neuromelanin in dopamine neurons shows an interesting link between aging and neurodegeneration.

Dopamine Oxidation and Autophagy

The molecular mechanisms involved in the neurodegenerative process of Parkinsons disease remain unclear. Currently, there is a general agreement that mitochondrial dysfunction, α-synuclein aggregation, oxidative stress, neuroinflammation, and impaired protein degradation are involved in the neurodegeneration of dopaminergic neurons containing neuromelanin in Parkinsons disease. Aminochrome has been proposed to play an essential role in the degeneration of dopaminergic neurons containing neuromelanin by inducing mitochondrial dysfunction, oxidative stress, the formation of neurotoxic α-synuclein protofibrils, and impaired protein degradation. Here, we discuss the relationship between the oxidation of dopamine to aminochrome, the precursor of neuromelanin, autophagy dysfunction in dopaminergic neurons containing neuromelanin, and the role of dopamine oxidation to aminochrome in autophagy dysfunction in dopaminergic neurons. Aminochrome induces the following: (i) the formation of α-synuclein protofibrils that inactivate chaperone-mediated autophagy; (ii) the formation of adducts with α- and β-tubulin, which induce the aggregation of the microtubules required for the fusion of autophagy vacuoles and lysosomes.

Stimulation of membrane serine-threonine phosphatase in erythrocytes by hydrogen peroxide and staurosporine

Indirect evidence has suggested that K-Cl cotransport in human and sheep erythrocytes is activated physiologically by a serine-threonine phosphatase. It is activated experimentally by H2O2 and by staurosporine, a kinase inhibitor. Activation by H2O2 and staurosporine is inhibited by serine-threonine phosphatase inhibitors, suggesting that the activators stimulate the phosphatase. The present study shows that sheep and human erythrocytes contain membrane-associated as well as cytosolic serine-threonine phosphatases, assayed from the dephosphorylation of 32P-labeled glycogen phosphorylase. In cells from both species, the relatively low sensitivity of the membrane enzyme to okadaic acid suggests it is type 1 protein phosphatase. The cytosolic phosphatase was much more sensitive to okadaic acid. Membrane-associated phosphatase was stimulated by both H2O2 and staurosporine. The results support earlier conclusions that the membrane-associated type 1 phosphatase identified here is regulated by phosphorylation and oxidation. The results are consistent with the phosphatase, or a portion of it, being responsible for activating K-Cl cotransport.

An oxidative stress‐mediated positive‐feedback iron uptake loop in neuronal cells

Intracellular reactive iron is a source of free radicals and a possible cause of cell damage. In this study, we analyzed the changes in iron homeostasis generated by iron accumulation in neuroblastoma (N2A) cells and hippocampal neurons. Increasing concentrations of iron in the culture medium elicited increasing amounts of intracellular iron and of the reactive iron pool. The cells had both IRP1 and IRP2 activities, being IRP1 activity quantitatively predominant. When iron in the culture medium increased from 1 to 40 µm, IRP2 activity decreased to nil. In contrast, IRP1 activity decreased when iron increased up to 20 µm, and then, unexpectedly, increased. IRP1 activity at iron concentrations above 20 µm was functional as it correlated with increased 55Fe uptake. The increase in IRP1 activity was mediated by oxidative‐stress as it was largely abolished by N‐acetyl‐l‐cysteine. Culturing cells with iron resulted in proteins and DNA modifications. In summary, iron uptake by N2A cells and hippocampus neurons did not shut off at high iron concentrations in the culture media. As a consequence, iron accumulated and generated oxidative damage. This behavior is probably a consequence of the paradoxical activation of IRP1 at high iron concentrations, a condition that may underlie some processes associated with neuronal degeneration and death.

Autophagy Protects Against Aminochrome-Induced Cell Death in Substantia Nigra-Derived Cell Line

Aminochrome, the precursor of neuromelanin, has been proposed to be involved in the neurodegeneration neuromelanin-containing dopaminergic neurons in Parkinsons disease. We aimed to study the mechanism of aminochrome-dependent cell death in a cell line derived from rat substantia nigra. We found that aminochrome (50μM), in the presence of NAD(P)H-quinone oxidoreductase, EC 1.6.99.2 (DT)-diaphorase inhibitor dicoumarol (DIC) (100μM), induces significant cell death (62 ± 3%; p < 0.01), increase in caspase-3 activation (p < 0.001), release of cytochrome C, disruption of mitochondrial membrane potential (p < 0.01), damage of mitochondrial DNA, damage of mitochondria determined with transmission electron microscopy, a dramatic morphological change characterized as cell shrinkage, and significant increase in number of autophagic vacuoles. To determine the role of autophagy on aminochrome-induced cell death, we incubated the cells in the presence of vinblastine and rapamycin. Interestingly, 10μM vinblastine induces a 5.9-fold (p < 0.001) and twofold (p < 0.01) significant increase in cell death when the cells were incubated with 30μM aminochrome in the absence and presence of DIC, respectively, whereas 10μM rapamycin preincubated 24 h before addition of 50μM aminochrome in the absence and the presence of 100μM DIC induces a significant decrease (p < 0.001) in cell death. In conclusion, autophagy seems to be an important protective mechanism against two different aminochrome-induced cell deaths that initially showed apoptotic features. The cell death induced by aminochrome when DT-diaphorase is inhibited requires activation of mitochondrial pathway, whereas the cell death induced by aminochrome alone requires inhibition of autophagy-dependent degrading of damaged organelles and recycling through lysosomes.

HFE inhibits apical iron uptake by intestinal epithelial (Caco-2) cells

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron metabolism characterized by increased intestinal iron absorption, which leads to progressive iron overload. The protein product of C282Y, the main mutation observed in HH, has lost the capacity to associate with β2‐microglobulin, preventing its targeting to the plasma membrane. The physiological mechanisms by which HFE, the normal product of the HH gene, regulates intestinal iron absorption are unknown. Under the hypothesis that HFE regulates intestinal iron absorption, we characterized the effect of HFE overexpression on apical iron uptake in intestinal epithelial Caco‐2 cells. We found that the primary effect of HFE overexpression was a marked reduction of apical iron uptake, despite a working iron responsive element/iron regulatory protein system and an eightfold increase in the mass of the iron transporter DMT1. The inhibitory effect of HFE on apical iron uptake reported here provides an explanation for the increased absorption of iron observed in HH, where the normal function of HFE is lost.

Aminochrome induces disruption of actin, alpha-, and beta-tubulin cytoskeleton networks in substantia-nigra-derived cell line.

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 μM aminochrome or 100 μM 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 μM aminochrome in the presence of 100 μM 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.

DT-Diaphorase Prevents Aminochrome-Induced Alpha-Synuclein Oligomer Formation and Neurotoxicity

It was reported that aminochrome induces the formation of alpha synuclein (SNCA) oligomers during dopamine oxidation. We found that DT-diaphorase (NQO1) prevents the formation of SNCA oligomers in the presence of aminochrome determined by Western blot, transmission electron microscopy, circular dichroism, and thioflavin T fluorescence, suggesting a protective role of NQO1 by preventing the formation of SNCA oligomers in dopaminergic neurons. In order to test NQO1 protective role in SNCA neurotoxicity in cellular model, we overexpressed SNCA in both RCSN-3 cells (wild-type) and RCSN-3Nq7 cells, which have constitutive expression of a siRNA against NQO1. The expression of SNCA in RCSN-3SNCA and RCSN-3Nq7SNCA cells increased 4.2- and 4.4-fold, respectively. The overexpression of SNCA in RCSN-3Nq7SNCA cells induces a significant increase in cell death of 2.8- and 3.2-fold when they were incubated with 50 and 70 µM aminochrome, respectively. The cell death was found to be of apoptotic character determined by annexin/propidium iodide technique with flow cytometry and DNA laddering. A Western blot demonstrated that SNCA in RCSN-3SNCA is only found in monomer form both in the presence of 20 µM aminochrome or cell culture medium contrasting with RCSN-3Nq7SNCA cells where the majority SNCA is found as oligomer. The antioligomer compound scyllo-inositol induced a significant decrease in aminochrome-induced cell death in RCSN-3Nq7SNCA cells in comparison to cells incubated in the absence of scyllo-inositol. Our results suggest that NQO1 seems to play an important role in the prevention of aminochrome-induced SNCA oligomer formation and SNCA oligomers neurotoxicity in dopaminergic neurons.

Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction

U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit 3H-dopamine uptake, which is inhibited by 2 µM of nomifensine and 15 µM of estradiol. We generated a stable cell line (U373MGsiGST6) expressing an siRNA against GSTM2 that resulted in low GSTM2 expression (26% of wild-type U373MG cells). A significant increase in cell death was observed when U373MGsiGST6 cells were incubated with 50 µM purified aminochrome (18-fold increase) compared with wild-type cells. The incubation of U373MGsiGST6 cells with 75 µM aminochrome resulted in the formation of autophagic vacuoles containing undigested cellular components, as determined using transmission electron microscopy. A significant increase in autophagosomes was determined by measuring endogenous LC3-II, a significant decrease in cell death was observed in the presence of bafilomycin A1, and a significant increase in cell death was observed in the presence of trehalose. A significant increase in LAMP2 immunostaining was observed, a significant decrease in bright red fluorescence of lysosomes with acridine orange was observed, and bafilomycin A1 pretreatment reduced the loss of lysosome acidity. A significant increase in cell death was observed in the presence of lysosomal protease inhibitors. Aggregation of TUBA/α-tubulin (tubulin, α) and SQSTM1 protein accumulation were also observed. Moreover, a significant increase in the number of lipids droplets was observed compared with U373MG cells with normal expression of GSTM2. These results support the notion that GSTM2 is a protective enzyme against aminochrome toxicity in astrocytes and that aminochrome cell death in U373MGsiGST6 cells involves autophagic-lysosomal dysfunction.