Carolina Perez-Pastene

Copper·Dopamine Complex Induces Mitochondrial Autophagy Preceding Caspase-independent Apoptotic Cell Death

Parkinsonism is one of the major neurological symptoms in Wilson disease, and young workers who worked in the copper smelting industry also developed Parkinsonism. We have reported the specific neurotoxic action of copper·dopamine complex in neurons with dopamine uptake. Copper·dopamine complex (100 μm) induces cell death in RCSN-3 cells by disrupting the cellular redox state, as demonstrated by a 1.9-fold increase in oxidized glutathione levels and a 56% cell death inhibition in the presence of 500 μm ascorbic acid; disruption of mitochondrial membrane potential with a spherical shape and well preserved morphology determined by transmission electron microscopy; inhibition (72%, p < 0.001) of phosphatidylserine externalization with 5 μm cyclosporine A; lack of caspase-3 activation; formation of autophagic vacuoles containing mitochondria after 2 h; transfection of cells with green fluorescent protein-light chain 3 plasmid showing that 68% of cells presented autophagosome vacuoles; colocalization of positive staining for green fluorescent protein-light chain 3 and Rhod-2AM, a selective indicator of mitochondrial calcium; and DNA laddering after 12-h incubation. These results suggest that the copper·dopamine complex induces mitochondrial autophagy followed by caspase-3-independent apoptotic cell death. However, a different cell death mechanism was observed when 100 μm copper·dopamine complex was incubated in the presence of 100 μm dicoumarol, an inhibitor of NAD(P)H quinone:oxidoreductase (EC 1.6.99.2, also known as DT-diaphorase and NQ01), because a more extensive and rapid cell death was observed. In addition, cyclosporine A had no effect on phosphatidylserine externalization, significant portions of compact chromatin were observed within a vacuolated nuclear membrane, DNA laddering was less pronounced, the mitochondria morphology was more affected, and the number of cells with autophagic vacuoles was a near 4-fold less.

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.

Monoamine transporter inhibitors and norepinephrine reduce dopamine-dependent iron toxicity in cells derived from the substantia nigra

The role of dopamine in iron uptake into catecholaminergic neurons, and dopamine oxidation to aminochrome and its one‐electron reduction in iron‐mediated neurotoxicity, was studied in RCSN‐3 cells, which express both tyrosine hydroxylase and monoamine transporters. The mean ± SD uptake of 100 µm59FeCl3 in RCSN‐3 cells was 25 ± 4 pmol per min per mg, which increased to 28 ± 8 pmol per min per mg when complexed with dopamine (Fe(III)–dopamine). This uptake was inhibited by 2 µm nomifensine (43%p < 0.05), 100 µm imipramine (62%p < 0.01), 30 µm reboxetine (71%p < 0.01) and 2 mm dopamine (84%p < 0.01). The uptake of 59Fe–dopamine complex was Na+, Cl– and temperature dependent. No toxic effects in RCSN‐3 cells were observed when the cells were incubated with 100 µm FeCl3 alone or complexed with dopamine. However, 100 µm Fe(III)–dopamine in the presence of 100 µm dicoumarol, an inhibitor of DT‐diaphorase, induced toxicity (44% cell death; p < 0.001), which was inhibited by 2 µm nomifensine, 30 µm reboxetine and 2 mm norepinephrine. The neuroprotective action of norepinephrine can be explained by (1) its ability to form complexes with Fe3+, (2) the uptake of Fe–norepinephrine complex via the norepinephrine transporter and (3) lack of toxicity of the Fe–norepinephrine complex even when DT‐diaphorase is inhibited. These results support the proposed neuroprotective role of DT‐diaphorase and norepinephrine.

Association of GST M1 null polymorphism with Parkinson's disease in a Chilean population with a strong Amerindian genetic component

We have studied the association of a null mutation of Glutathione Transferase M1 (GST M1*0/0) with Parkinsons disease (MIM 168600) in a Chilean population with a strong Amerindian genetic component. We determined the genotype in 349 patients with idiopathic Parkinsons disease (174 female and 175 male; 66.84+/-10.7 years of age), and compared that to 611 controls (457 female and 254 male; 62+/-13.4 years of age). A significant association of the null mutation in GST M1 with Parkinsons disease was found (p=0.021), and the association was strongest in the earlier age range. An association of GSTM1*0/0 with Parkinsons disease supports the hypothesis that Glutathione Transferase M1 plays a role in protecting astrocytes against toxic dopamine oxidative metabolism, and most likely by preventing toxic one-electron reduction of aminochrome.

Aminochrome as a preclinical experimental model to study degeneration of dopaminergic neurons in Parkinson’s disease

Four decades after L-dopa introduction to PD therapy, the cause of Parkinson’s disease (PD) remains unknown despite the intensive research and the discovery of a number of gene mutations and delections in the pathogenesis of familial PD. Different model neurotoxins have been used as preclinical experimental models to study the neurodegenerative process in PD, such as 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and rotenone. The lack of success in identifying the molecular mechanism for the degenerative process in PD opens the question whether the current preclinical experimental models are suitable to understand the degeneration of neuromelanin-containing dopaminergic neurons in PD. We propose aminochrome as a model neurotoxin to study the neurodegenerative processes occurring in neuromelanin-containing dopaminergic neurons in PD. Aminochrome is an endogenous compound formed during dopamine oxidation and it is the precursor of neuromelanin, a substance whose formation is a normal process in mesencephalic dopaminergic neurons. However, aminochrome itself can induce neurotoxicity under certain aberrant conditions such as (i) one-electron reduction of aminochrome catalyzed by flavoenzymes to leukoaminochrome-o-semiquinone radical, which is a highly reactive neurotoxin; or (ii) the formation of aminochrome adducts with alpha-synuclein, enhancing and stabilizing the formation of neurotoxic protofibrils. These two neurotoxic pathways of aminochrome are prevented by DT-diaphorase, an enzyme that effectively reduces aminochrome with two-electrons, preventing both aminochrome one-electron reduction or formation alpha-synuclein protofibrils. We propose to use aminochrome as a preclinical experimental model to study the neurodegenerative process of neuromelanin-containing dopaminergic neurons in PD.

Molecular and neurochemical mechanisms in PD pathogenesis.

Oxidation of dopamine to aminochrome seems to be a normal process leading to aminochrome polymerization to form neuromelanin, since normal individuals have this pigment in their dopaminergic neurons in the substantia nigra. The neurons lost in individuals with Parkinson’s disease are dopaminergic neurons containing neuromelanin. This raises two questions. First, why are those cells containing neuromelanin lost in this disease? Second, what is the identity of the neurotoxin that induces this cell death? We propose that aminochrome is the agent responsible for the death of dopaminergic neurons containing neuromelanin in individuals with Parkinson’s disease. The normal oxidative pathway of dopamine, in which aminochrome polymerizes to form neuromelanin, can be neurotoxic if DT-diaphorase is inhibited under certain conditions. Inhibition of DT-diaphorase allows two neurotoxic reactions to proceed: (i) the formation of aminochrome adducts with alpha-synuclein, which induce and stabilize the formation of neurotoxic protofibrils; and (ii) the one electron reduction of aminochrome to the neurotoxic leukoaminochrome o-semiquinone radical. Therefore, we propose that DT-diaphorase is an important neuroprotective enzyme in dopaminergic neurons containing neuromelanin.

Lrrk2 mutations in South America : A study of Chilean Parkinson's disease

Pathogenic substitutions in the leucine-rich repeat kinase 2 protein (Lrrk2), R1441G and G2019S, are a prevalent cause of autosomal dominant and sporadic Parkinsons disease in the Northern Spanish population. In this study we examined the frequency of these two substitutions in 166 Parkinsons disease patients and 153 controls from Chile, a population with Spanish/European-Amerindian admixture. Lrrk2 R1441G was not observed, however Lrrk2 G2019S was detected in one familial and four sporadic Parkinsons disease patients. These findings suggest Lrrk2 G2019S may play an important role in Parkinsons disease on the South American Continent and further studies are now warranted.

The catecholaminergic RCSN-3 cell line: A model to study dopamine metabolism

RCSN-3 cells are a cloned cell line derived from the substantia nigra of an adult rat. The cell line grows in monolayer and does not require differentiation to express catecholaminergic traits, such as (i) tyrosine hydroxylase; (ii) dopamine release; (iii) dopamine transport; (iv) norepinephrine transport; (v) monoamine oxidase (MAO)-A expression, but not MAO-B; (vi) formation of neuromelanin; (vii) vesicular monoamine transporter-2 (VMAT-2) expression. In addition, this cell line expresses serotonin transporters, divalent metal transporter, DMT1, dopamine receptor 1 mRNA under proliferating conditions, and dopamine receptor 5 mRNA after incubation with dopamine or dicoumarol. Expression of dopamine receptors D2, D3 and D4 mRNA were not detected in proliferating cells or when the cells were treated with dopamine, CuSO4, dicoumarol or dopamine-copper complex. Angiotensin II receptor mRNA was also found to be expressed, but it underwent down regulation in the presence of aminochrome. Total quinone reductase activity corresponded 94% to DT-diaphorase. The cells also express antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase. This cell line is a suitablein vitro model for studies of dopamine metabolism, since under proliferating conditions the cells express all the pertinent markers.