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[This corrects the article on p. 161 in vol. 10, PMID: 27147953.].
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Astrocytes are exposed to aminochrome via the oxidation of dopamine that is taken up from the synaptic cleft after its release from dopaminergic neurons. Glutathione transferase M2-2 (GSTM2) has been shown to protect astrocytes from aminochrome-induced toxicity, but astrocytes also express DT-diaphorase, which has been shown to prevent aminochrome-induced neurotoxicity in dopaminergic neurons. Therefore, the question is whether DT-diaphorase also protects astrocytes from aminochrome-induced toxicity. DT-diaphorase is constitutively expressed in U373MG cells, and its inhibition by dicoumarol induced a significant increase of aminochrome-induced cell death. However, the inhibition of DT-diaphorase in U373MGsiGST6 cells, which have 74% of GSTM2 gene expression silenced, resulted in a more than 2-fold increase in cell death, suggesting that DT-diaphorase plays an important role in preventing aminochrome-induced toxicity in astrocytes.
Assuntos
Astrócitos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Indolquinonas/toxicidade , NAD(P)H Desidrogenase (Quinona)/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Transformada , Dicumarol/farmacologia , Inibidores Enzimáticos/farmacologia , Glutationa Transferase/metabolismo , Humanos , RNA Interferente Pequeno/metabolismoRESUMO
Neurodegenerative disorders have a common characteristic that is the involvement of different cell types, typically the reactivity of astrocytes and microglia, characterizing gliosis, which in turn contributes to the neuronal dysfunction and or death. Flavonoids are secondary metabolites of plant origin widely investigated at present and represent one of the most important and diversified among natural products phenolic groups. Several biological activities are attributed to this class of polyphenols, such as antitumor activity, antioxidant, antiviral, and anti-inflammatory, among others, which give significant pharmacological importance. Our group have observed that flavonoids derived from Brazilian plants Dimorphandra mollis Bent., Croton betulaster Müll. Arg., e Poincianella pyramidalis Tul., botanical synonymous Caesalpinia pyramidalis Tul. also elicit a broad spectrum of responses in astrocytes and neurons in culture as activation of astrocytes and microglia, astrocyte associated protection of neuronal progenitor cells, neuronal differentiation and neuritogenesis. It was observed the flavonoids also induced neuronal differentiation of mouse embryonic stem cells and human pluripotent stem cells. Moreover, with the objective of seeking preclinical pharmacological evidence of these molecules, in order to assess its future use in the treatment of neurodegenerative disorders, we have evaluated the effects of flavonoids in preclinical in vitro models of neuroinflammation associated with Parkinson's disease and glutamate toxicity associated with ischemia. In particular, our efforts have been directed to identify mechanisms involved in the changes in viability, morphology, and glial cell function induced by flavonoids in cultures of glial cells and neuronal cells alone or in interactions and clarify the relation with their neuroprotective and morphogetic effects.
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Flavonoides/farmacologia , Flavonoides/uso terapêutico , Doenças Neurodegenerativas/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Plantas/química , Animais , Células Cultivadas , Humanos , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêuticoRESUMO
L-Dopa continues to be the gold drug in Parkinson's disease (PD) treatment from 1967. The failure to translate successful results from preclinical to clinical studies can be explained by the use of preclinical models which do not reflect what happens in the disease since these induce a rapid and extensive degeneration; for example, MPTP induces a severe Parkinsonism in only 3 days in humans contrasting with the slow degeneration and progression of PD. This study presents a new anatomy and develops preclinical model based on aminochrome which induces a slow and progressive dysfunction of dopaminergic neurons. The unilateral injection of aminochrome into rat striatum resulted in (1) contralateral rotation when the animals are stimulated with apomorphine; (2) absence of significant loss of tyrosine hydroxylase-positive neuronal elements both in substantia nigra and striatum; (3) cell shrinkage; (4) significant reduction of dopamine release; (5) significant increase in GABA release; (6) significant decrease in the number of monoaminergic presynaptic vesicles; (7) significant increase of dopamine concentration inside of monoaminergic vesicles; (8) significant increase of damaged mitochondria; (9) significant decrease of ATP level in the striatum (10) significant decrease in basal and maximal mitochondrial respiration. These results suggest that aminochrome induces dysfunction of dopaminergic neurons where the contralateral behavior can be explained by aminochrome-induced ATP decrease required both for anterograde transport of synaptic vesicles and dopamine release. Aminochrome could be implemented as a new model neurotoxin to study Parkinson's disease.
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Neurônios Dopaminérgicos/efeitos dos fármacos , Indolquinonas/farmacologia , Doença de Parkinson/patologia , Trifosfato de Adenosina/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Dopamina/análise , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Humanos , Indolquinonas/síntese química , Indolquinonas/química , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/fisiologia , Doença de Parkinson/metabolismo , Doença de Parkinson/veterinária , Ratos , Ratos Sprague-Dawley , Substância Negra/metabolismo , Vesículas Sinápticas/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Ácido gama-Aminobutírico/análiseRESUMO
In this study, we investigated the role of adducts formation between aminochrome and tubulin and its interference in microtubules assembly and stability in aminochrome-induced toxicity in SH-SY5Y cells. We also investigated whether changes in the microtubules structures are an early event that could affect tubulin expression. We demonstrated in vitro that aminochrome tubulin adducts inhibit tubulin polymerization and that aminochrome induces microtubules disassembly. Moreover, when the SH-SY5Y cells were incubated with aminochrome, we observed an increase in soluble tubulin, indicating depolymerization of microtubules. Aminochrome generates disruption of the microtubules network, leading to changes in the morphology of the cells inducing cell death, in a dose- and time-dependent manner. Interestingly, these changes preceded cell death and were partly inhibited by paclitaxel, a microtubule-stabilizing agent. Furthermore, we observed that aminochrome increased early tubulin expression before significant cell death occurred. Consequently, all these antecedents suggest that aminochrome toxicity is mediated by early disruption of microtubules network, where the adduct formation between aminochrome and tubulin could be responsible for the inhibition in the assembly microtubules and the loss of microtubules stability. Possibly, the early changes in tubulin expression could correspond to compensatory mechanisms against the toxic effects of aminochrome.
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Indolquinonas/toxicidade , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Moduladores de Tubulina/toxicidade , Tubulina (Proteína)/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , HumanosRESUMO
The pharmacological treatment of Parkinson's disease (PD) is limited to dopamine agonists and anti-cholinergic drugs that do not stop the progress of disease. LDopa was introduced to the treatment in 1967; this drug is still the best and most commonly used drug since it generates a real improvement in patient quality of life, but the disadvantage of L-dopa is that this positive effect is followed by severe side effects such as dyskinesia. The search for a new drug in the treatment of PD is limited to compounds which decrease the side effects of the drugs used in the treatment of the disease, such as L-dopa-induced dyskinesia. One possible explanation for pharmaceutical companies not developing new drugs to stop disease development is because the mechanism which induces the loss of dopaminergic neurons containing neuromelanin of the nigrostriatal system is still unknown. The discovery of genes (alpha-synuclein, parkin, pink-1, DJ- 1, LRRK2, GBA1, etc.) associated with familial forms of PD resulted in an enormous input into basic research in order to understand the role of these proteins in the disease. It is generally accepted that the loss of dopaminergic neurons containing neuromelanin involves mitochondrial dysfunction, protein degradation dysfunction, the aggregation of alpha-synuclein to neurotoxic oligomers, oxidative neuroinflammation and endoplasmic reticulum stress, but the question of what induces these mechanisms remains unanswered. Aminochrome, the product of dopamine oxidation and the precursor of neuromelanin, is directly involved in five of the six mechanisms and may be a better PD preclinical model.
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Descoberta de Drogas/métodos , Indolquinonas/farmacologia , Doença de Parkinson/prevenção & controle , Animais , Dopamina/metabolismo , Humanos , Indolquinonas/química , Oxirredução , Doença de Parkinson/metabolismoRESUMO
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.
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Benzopiranos/toxicidade , Biopolímeros/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Sistema Nervoso/efeitos dos fármacos , alfa-Sinucleína/metabolismo , Linhagem Celular , HumanosRESUMO
U373MG cells are able to take up aminochrome that induces glutathione transferase M2-2 (GSTM2) expression in a concentration-dependent manner where 100 µM aminochrome increases GSTM2 expression by 2.1-fold (P < 0.001) at 3 h. The uptake of (3)H-aminochrome into U373MG cells was significantly reduced in the presence of 2 µM nomifensine (P < 0.001) 100 µM imipramine (P < 0.001) and 50 mM dopamine (P < 0.001). Interestingly, U373MG cells excrete GSTM2 into the conditioned medium and the excretion was significantly increased (2.7-fold; P < 0.001) when the cells were pretreated with 50 µM aminochrome for 3 h. The U373MG-conditioned medium containing GSTM2 protects SH-SY5Y cells incubated with 10 µM aminochrome. The significant protection provided by U373MG-conditioned medium in SH-SY5Y cells incubated with aminochrome was dependent on GSTM2 internalization into SH-SY5Y cells as evidenced by (i) uptake of (14)C-GSTM2 released from U373MG cells into SH-SY5Y cells, a process inhibited by anti-GSTM2 antiserum; (ii) lack of protection of U373MG-conditioned medium in the presence of anti-GSTM2 antiserum on SH-SY5Y cells treated with aminochrome; and (iii) lack of protection of conditioned medium from U373MGsiGST6 that expresses an siRNA directed against GSTM2 on SH-SY5Y cells treated with aminochrome. In conclusion, our results demonstrated that U373MG cells protect SH-SY5Y cells against aminochrome neurotoxicity by releasing GSTM2 into the conditioned medium and subsequent internalization of GSTM2 into SH-SY5Y cells. These results suggest a new mechanism of protection of dopaminergic neurons mediated by astrocytes by releasing GSTM2 into the intersynaptic space and subsequent internalization into dopaminergic neuron in order to protect these cells against aminochrome neurotoxicity.