RESUMO

Investigations of the effect of antioxidants on idiopathic Parkinson's disease have been unsuccessful because the preclinical models used to propose these clinical studies do not accurately represent the neurodegenerative process of the disease. Treatment with certain exogenous neurotoxins induces massive and extremely rapid degeneration; for example, MPTP causes severe Parkinsonism in just three days, while the degenerative process of idiopathic Parkinson´s disease proceeds over many years. The endogenous neurotoxin aminochrome seems to be a good alternative target since it is formed in the nigrostriatal system neurons where the degenerative process occurs. Aminochrome induces all the mechanisms reported to be involved in the degenerative processes of idiopathic Parkinson's disease. The presence of neuromelanin-containing dopaminergic neurons in the postmortem brain of healthy elderly people suggests that neuromelanin synthesis is a normal and harmless process despite the fact that it requires oxidation of dopamine to three ortho-quinones that are potentially toxic, especially aminochrome. The apparent contradiction that neuromelanin synthesis is harmless, despite its formation via neurotoxic ortho-quinones, can be explained by the protective roles of DT-diaphorase and glutathione transferase GSTM2-2 as well as the neuroprotective role of astrocytes secreting exosomes loaded with GSTM2-2. Increasing the expression of DT-diaphorase and GSTM2-2 may be a therapeutic goal to prevent the degeneration of new neuromelanin-containing dopaminergic neurons. Several phytochemicals that induce DT-diaphorase have been discovered and, therefore, an interesting question is whether these phytochemical KEAP1/NRF2 activators can inhibit or decrease aminochrome-induced neurotoxicity.

RESUMO

Glutathione is an important antioxidant that plays a crucial role in the cellular protection against oxidative stress and detoxification of electrophilic mutagens, and carcinogens. Glutathione transferases are enzymes catalyzing glutathione-dependent reactions that lead to inactivation and conjugation of toxic compounds, processes followed by subsequent excretion of the detoxified products. Degeneration and loss of neuromelanin-containing dopaminergic neurons in the nigrostriatal neurons generally involves oxidative stress, neuroinflammation, alpha-synuclein aggregation to neurotoxic oligomers, mitochondrial dysfunction, protein degradation dysfunction, and endoplasmic reticulum stress. However, it is still unclear what triggers these neurodegenerative processes. It has been reported that aminochrome may elicit all of these mechanisms and, interestingly, aminochrome is formed inside neuromelanin-containing dopaminergic neurons during neuromelanin synthesis. Aminochrome is a neurotoxic ortho-quinone formed in neuromelanin synthesis. However, it seems paradoxical that the neurotoxin aminochrome is generated during neuromelanin synthesis, even though healthy seniors have these neurons intact when they die. The explanation of this paradox is the existence of protective tools against aminochrome neurotoxicity composed of the enzymes DT-diaphorase, expressed in these neurons, and glutathione transferase M2-2, expressed in astrocytes. Recently, it has been reported that dopaminergic neurons can be protected by glutathione transferase M2-2 from astrocytes, which secrete exosomes containing the protective enzyme.

RESUMO

Astrocytes protect neurons by modulating neuronal function and survival. Astrocytes support neurons in several ways. They provide energy through the astrocyte-neuron lactate shuttle, protect neurons from excitotoxicity, and internalize neuronal lipid droplets to degrade fatty acids for neuronal metabolic and synaptic support, as well as by their high capacity for glutamate uptake and the conversion of glutamate to glutamine. A recent reported astrocyte system for protection of dopamine neurons against the neurotoxic products of dopamine, such as aminochrome and other o-quinones, were generated under neuromelanin synthesis by oxidizing dopamine catechol structure. Astrocytes secrete glutathione transferase M2-2 through exosomes that transport this enzyme into dopaminergic neurons to protect these neurons against aminochrome neurotoxicity. The role of this new astrocyte protective mechanism in Parkinson´s disease is discussed.

RESUMO

The enzyme glutathione transferase M2-2, expressed in human astrocytes, increases its expression in the presence of aminochrome and catalyzes the conjugation of aminochrome, preventing its toxic effects. Secretion of the enzyme glutathione transferase M2-2 from U373MG cells, used as a cellular model for astrocytes, has been reported, and the enzyme is taken up by neuroblastoma SYSH-S7 cells and provide protection against aminochrome. The present study provides evidence that glutathione transferase M2-2 is released in exosomes from U373MG cells, thereby providing a means for intercellular transport of the enzyme. With particular relevance to Parkinson disease and other degenerative conditions, we propose a new mechanism by which astrocytes may protect dopaminergic neurons against the endogenous neurotoxin aminochrome.

Assuntos

RESUMO

Aminochrome induces neurotoxic alpha-synuclein oligomer formation relevant to the etiology of Parkinson's disease. Oxidative stress produces aminochrome from dopamine, but conjugation with glutathione catalyzed by glutathione transferase M2-2 significantly decreases aminochrome-induced toxicity and alpha-synuclein oligomer formation. Notably, in the presence of the aminochrome-glutathione conjugate, previously unknown species of alpha-synuclein oligomers are formed. These aminochrome-glutathione oligomers of alpha-synuclein differ from formerly characterized oligomers and (i) have high molecular weight, and are stable and SDS-resistant, as determined by the Western blot method, (ii) show positive NBT-quinone-protein staining, which indicates the formation of alpha-synuclein adducts containing aminochrome. Furthermore, aminochrome-glutathione alpha-synuclein oligomers (iii) have distinctive shape and size, as determined by transmission electron microscopy, and (iv) are not toxic in U373MG cells. In conclusion, glutathione conjugated with aminochrome induces a new type of alpha-synuclein oligomers of a different size and shape, which have no demonstrable toxicity.

Assuntos

RESUMO

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.

Assuntos

RESUMO

U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit (3)H-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 A 1, 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 A 1 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.

Assuntos