Eduardo Couve

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.

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.

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.

Changes in the Predentin Thickness and Mineralization Front Configuration in Developing Human Premolars

The thickness of the predentin layer was studied at three different levels of developing human premolars. The results demonstrate that at the growing end next to the apex, where dentinogenesis is most active, predentin exhibits its greatest thickness (mean value 40.4 micron). However, at the coronal region, where primary dentin has been completely formed, predentin width is reduced to a mean value of 14.8 micron. Changes in the calcospheritic configuration of the mineralization front were established for each of the predentin levels studied. A comparative analysis of these calcospheritic changes and the morphology of fluorescent tetracycline lines detected in ground sections of premolars was established. Fluorescent lines observed at the coronal circumpulpal dentin showed large calcospheritic forms beneath the mantle dentin. However, lines found near the dentin-pulp border showed small calcospherites. It is concluded that the thickness of the predentin layer and the mineralization front configuration vary as a function of dentinogenic activity during development of human premolars.

Cell lines derived from hippocampal neurons of the normal and trisomy 16 mouse fetus (a model for Down syndrome) exhibit neuronal markers, cholinergic function, and functional neurotransmitter receptors.

We have established hippocampal cell lines from normal and trisomy 16 fetal mice, a model of human trisomy 21. Both cell lines, named H1b (derived from a normal animal) and HTk (trisomic) possess neuronal markers by immunohistochemistry (enolase, synaptophysin, microtubule associated protein-2, and choline acetyltransferase) and lack glial markers (glial fibrillary acidic protein and S-100). Also, we evaluated intracellular Ca(2+) levels ([Ca(2+)](i)) in response to neurotransmitter agonists, in cells loaded with the fluorescent Ca(2+) indicators Indo-1 and Fluo-3. Both cell lines responded to glutamatergic stimuli induced by glutamate, N-methyl-D-aspartate, I-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazole propanoic acid or kainate. Glutamate responses were only partially prevented by addition of 5 mM EGTA and the metabotropic glutamate receptor agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD), increased [Ca(2+)](i) in both cell types. These results confirm the presence of glutamatergic metabotropic receptors. In glutamate-induced responses, HTk cells exhibited slower time-dependent decay kinetics than H1b cells. Cholinergic agonists (nicotine and muscarine) induced a rapid, transient increase in [Ca(2+)](i) in both cell types. Furthermore, some cells were sensitive to histamine and norepinephrine. All responses to the aforementioned agonists were prevented by addition of specific antagonists. We also studied incorporation and release of [(3)H]choline in the cells, and observed no differences in uptake parameters. However, release induced by K(+) and nicotine depolarization was greatly reduced in HTk cells. The results show that H1b and HTk cells retain neuronal characteristics and respond to specific neurotransmitter stimuli. The HTk differences could be related to neuronal pathophysiology in Down syndrome.