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- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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- Cuevas, Carlos, et al.
(författare)
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Glutathione Transferase-M2-2 Secreted from Glioblastoma Cell Protects SH-SY5Y Cells from Aminochrome Neurotoxicity
- 2015
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Ingår i: Neurotoxicity research. - : Springer Science and Business Media LLC. - 1029-8428 .- 1476-3524. ; 27:3, s. 217-228
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Tidskriftsartikel (refereegranskat)abstract
- U373MG cells are able to take up aminochrome that induces glutathione transferase M2-2 (GSTM2) expression in a concentration-dependent manner where 100 A mu M aminochrome increases GSTM2 expression by 2.1-fold (P < 0.001) at 3 h. The uptake of H-3-aminochrome into U373MG cells was significantly reduced in the presence of 2 A mu M nomifensine (P < 0.001) 100 A mu M 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 A mu M aminochrome for 3 h. The U373MG-conditioned medium containing GSTM2 protects SH-SY5Y cells incubated with 10 A mu M 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 C-14-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.
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| 5. |
- Huenchuguala, Sandro, et al.
(författare)
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Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction
- 2014
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8627 .- 1554-8635. ; 10:4, s. 618-630
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Tidskriftsartikel (refereegranskat)abstract
- U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit H-3-dopamine uptake, which is inhibited by 2 mu M of nomifensine and 15 mu M 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 mu M purified aminochrome (18-fold increase) compared with wild-type cells. The incubation of U373MGsiGST6 cells with 75 mu M 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/-tubulin (tubulin, ) 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.
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| 6. |
- Huenchuguala, Sandro, et al.
(författare)
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Novel Alpha-Synuclein Oligomers Formed with the Aminochrome-Glutathione Conjugate Are Not Neurotoxic
- 2019
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Ingår i: Neurotoxicity research. - : Springer Science and Business Media LLC. - 1029-8428 .- 1476-3524. ; 35:2, s. 432-440
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Segura-Aguilar, Juan, et al.
(författare)
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A Preclinical Model for Parkinson's Disease Based on Transcriptional Gene Activation via KEAP1/NRF2 to Develop New Antioxidant Therapies
- 2023
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Ingår i: Antioxidants. - : MDPI AG. - 2076-3921. ; 12:3
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Forskningsöversikt (refereegranskat)abstract
- 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.
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| 8. |
- Segura-Aguilar, Juan, et al.
(författare)
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Astrocytes protect dopaminergic neurons against aminochrome neurotoxicity
- 2022
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Ingår i: Neural Regeneration Research. - : Medknow. - 1673-5374 .- 1876-7958. ; 17:9, s. 1861-1866
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Forskningsöversikt (refereegranskat)abstract
- 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.
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