A Bibliometric Evaluation of the Top 100 Cited Dimethyl Fumarate Articles.

Dimethyl fumarate is a cytoprotective and immunomodulatory drug used in the treatment of multiple sclerosis. We performed a bibliometric study examining the characteristics and trends of the top 100 cited articles that include dimethyl fumarate in the title. On 21 September 2020 we carried out an electronic search in the Web of Science (WOS), seeking articles that include the following terms within the title: dimethyl fumarate, BG-12, or Tecfidera. To focus our investigation on original research, we refined the search to include only articles, early access, others, case report, and clinical trials. We obtained a total of 1115 items, which were cited 7169 times, had a citation density of 6.43 citations/item, and an h-index of 40. Around 2010, there was a jump in the number of published articles per year, rising from 5 articles/year up to 12 articles/year. We sorted all the items by the number of citations and selected the top 100 most cited (T100). The T100 had 4164 citations, with a density of 37 citations/year and contained 16 classic research articles. They were published between 1961 and 2018; the years 2010-2018 amassed nearly 80% of the T100. We noted 17 research areas with articles in the T100. Of these, the number one ranking went to neurosciences/neurology with 39 articles, and chemistry ranked second on the T100 list with 14 items. We noticed that the percentage of articles belonging to different journals changed depending on the time period. Chemistry held the highest number of papers during 1961-2000, while pharmacology andneurosciences/neurology led the 2001-2018 interval. A total of 478 authors from 145 institutions and 25 countries were included in the T100 ranking. The paper by Gold R et al. was the most successful with 14 articles, 1.823 citations and a density of 140.23 citations/year. The biotechnological company Biogen led the T100 list with 20 articles. With 59 published articles, the USA was the leading country in publications. We concluded that this study analyzed the use of and research on dimethyl fumarate from a different perspective, which will allow the readership (expert or not) to understand the relevance of classic and recent literature on this topic.

Bcl-xL-mediated antioxidant function abrogates the disruption of mitochondrial dynamics induced by LRRK2 inhibition.

We have used the human neuroblastoma cell line SH-SY5Y overexpressing Bcl-xL (SH-SY5Y/Bcl-xL) to clarify the effects of this mitochondrial protein on the control of mitochondrial dynamics and the autophagic processes which occur after the inhibition of leucine-rich repeat kinase 2 (LRRK2) with GSK2578215A. In wild type (SH-SY5Y/Neo) cells, GSK2578215A (1nM) caused a disruption of mitochondrial morphology and an imbalance in intracellular reactive oxygen species (ROS) as indicated by an increase in dichlorofluorescein fluorescence and 4-hydroxynonenal. However, SH-SY5Y/Bcl-xL cells under GSK2578215A treatment, unlike the wild type, preserved a high mitochondrial membrane potential and did not exhibit apoptotical chromatins. In contrast to wild type cells, in SH-SY5Y/Bcl-xL cells, GSK2578215A did not induce mitochondrial translocation of neither dynamin related protein-1 nor the proapoptotic protein, Bax. In SH-SY5Y/Neo, but not SH-SY5Y/Bcl-xL cells, mitochondrial fragmentation elicited by GSK2578215A precedes an autophagic response. Furthermore, the overexpression of Bcl-xL protein restores the autophagic flux pathway disrupted by this inhibitor. SH-SY5Y/Neo, but not SH-SY5Y/Bcl-xL cells, responded to LRRK2 inhibition by an increase in the levels of acetylated tubulin, indicating that this was abrogated by Bcl-xL overexpression. This hyperacetylation of tubulin took place earlier than any of the above-mentioned events suggesting that it is involved in the autophagic flux interruption. Pre-treatment with tempol prevented the GSK2578215A-induced mitochondrial fragmentation, autophagy and the rise in acetylated tubulin in SH-SY5Y/Neo cells. Thus, these data support the notion that ROS act as a second messenger connexion between LRRK2 inhibition and these deleterious responses, which are markedly alleviated by the Bcl-xL-mediated ROS generation blockade.

On the mechanism underlying ethanol-induced mitochondrial dynamic disruption and autophagy response.

We have explored the mechanisms underlying ethanol-induced mitochondrial dynamics disruption and mitophagy. Ethanol increases mitochondrial fission in a concentration-dependent manner through Drp1 mitochondrial translocation and OPA1 proteolytic cleavage. ARPE-19 (a human retinal pigment epithelial cell line) cells challenged with ethanol showed mitochondrial potential disruptions mediated by alterations in mitochondrial complex IV protein level and increases in mitochondrial reactive oxygen species production. In addition, ethanol activated the canonical autophagic pathway, as denoted by autophagosome formation and autophagy regulator elements including Beclin1, ATG5-ATG12 and P-S6 kinase. Likewise, autophagy inhibition dramatically increased mitochondrial fission and cell death, whereas autophagy stimulation rendered the opposite results, placing autophagy as a cytoprotective response aimed to remove damaged mitochondria. Interestingly, although ethanol induced mitochondrial Bax translocation, this episode was associated to cell death rather than mitochondrial fission or autophagy responses. Thus, Bax required 600 mM ethanol to migrate to mitochondria, a concentration that resulted in cell death. Furthermore, mouse embryonic fibroblasts lacking this protein respond to ethanol by undergoing mitochondrial fission and autophagy but not cytotoxicity. Finally, by using the specific mitochondrial-targeted scavenger MitoQ, we revealed mitochondria as the main source of reactive oxygen species that trigger autophagy activation. These findings suggest that cells respond to ethanol activating mitochondrial fission machinery by Drp1 and OPA1 rather than bax, in a manner that stimulates cytoprotective autophagy through mitochondrial ROS.

The mitochondria-targeted anti-oxidant MitoQ reduces aspects of mitochondrial fission in the 6-OHDA cell model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder for which available treatments provide symptom relief but do not stop disease progression. Mitochondria, and in particular mitochondrial dynamics, have been postulated as plausible pharmacological targets. Mitochondria-targeted antioxidants have been developed to prevent mitochondrial oxidative damage, and to alter the involvement of reactive oxygen species (ROS) in signaling pathways. In this study, we have dissected the effect of MitoQ, which is produced by covalent attachment of ubiquinone to a triphenylphosphonium lipophilic cation by a ten carbon alkyl chain. MitoQ was tested in an in vitro PD model which involves addition of 6-hydroxydopamine (6-OHDA) to SH-SY5Y cell cultures. At sublethal concentrations of 50µM, 6-OHDA did not induce increases in protein carbonyl, mitochondrial lipid peroxidation or mitochondrial DNA damage. However, after 3h of treatment, 6-OHDA disrupts the mitochondrial morphology and activates the machinery of mitochondrial fission, but not fusion. Addition of 6-OHDA did not increase the levels of fission 1, mitofusins 1 and 2 or optic atrophy 1 proteins, but does lead to the translocation of dynamin related protein 1 from the cytosol to the mitochondria. Pre-treatment with MitoQ (50nM, 30min) results in the inhibition of the mitochondrial translocation of Drp1. Furthermore, MitoQ also inhibited the translocation of the pro-apoptotic protein Bax to the mitochondria. These findings provide mechanistic evidence for a role for redox events contributing to mitochondrial fission and suggest the potential of mitochondria-targeted therapeutics in diseases that involve mitochondrial fragmentation due to oxidative stress.

Vitamin D Receptor Polymorphisms in a Spanish Cohort of Parkinson's Disease Patients.

Background: Vitamin D receptor (VDR) is a nuclear hormone receptor widely expressed in the substantia nigra. Its association with an increased risk of Parkinson's disease (PD) is based on vitamin D deficiency and/or different polymorphisms in its gene receptor. This fact has been demonstrated by several case-control studies. Materials and Methods: Consequently, we investigated the association between VDR ApaI, BsmI, FokI, and TaqI gene polymorphisms and PD in a Spanish cohort that included 54 cases and 17 healthy controls. The detection of single nucleotide polymorphisms (SNPs) was performed using a polymerase chain reaction-restriction fragment length polymorphism. Results: Our data indicate that the SNPs were not associated with the age of onset of PD, nor with the occurrence of motor symptoms. However, only BsmI polymorphism was significantly associated with PD in this Spanish cohort. In fact, BsmI genotype was five times higher among PD patients than among controls, and the A allele was considered as a genetic risk for PD. Additionally, the combination of FokI and BsmI polymorphisms was significantly associated with PD and could represent a risk factor. Conclusion: We conclude that ApaI, TaqI, and FokI polymorphisms were not associated with PD, but BsmI could be a risk factor for PD in this randomized population.

Comparative Genetic Analysis of the Promoters of the ATG16L1 and ATG5 Genes Associated with Sporadic Parkinson's Disease.

Sporadic Parkinson's disease, characterised by a decline in dopamine, usually manifests in people over 65 years of age. Although 10% of cases have a genetic (familial) basis, most PD is sporadic. Genome sequencing studies have associated several genetic variants with sporadic PD. Our aim was to analyse the promoter region of the ATG16L1 and ATG5 genes in sporadic PD patients and ethnically matched controls. Genotypes were obtained by using the Sanger method with primers designed by us. The number of haplotypes was estimated with DnaSP software, phylogeny was reconstructed in Network, and genetic divergence was explored with Fst. Seven and two haplotypes were obtained for ATG16L1 and ATG5, respectively. However, only ATG16L1 showed a significant contribution to PD and a significant excess of accumulated mutations that could influence sporadic PD disease. Of a total of seven haplotypes found, only four were unique to patients sharing the T allele (rs77820970). Recent studies using MAPT genes support the notion that the architecture of haplotypes is worthy of being considered genetically risky, as shown in our study, confirming that large-scale assessment in different populations could be relevant to understanding the role of population-specific heterogeneity. Finally, our data suggest that the architecture of certain haplotypes and ethnicity determine the risk of PD, linking haplotype variation and neurodegenerative processes.

Minocycline exerts uncoupling and inhibiting effects on mitochondrial respiration through adenine nucleotide translocase inhibition.

The present study was aimed to provide a better understanding of the mitochondria-targeted actions of minocycline (MC), a second-generation tetracycline which has cytoprotective effects. Although the specific mechanisms underlying its activity remained elusive, considerable amounts of data indicated mitochondria as the primary pharmacological target of MC. Previous reports have shown that MC affects the oxygen-uptake rate by isolated mitochondria in different respiratory states. Here, we report on the effect of MC, in the range 50-200µM, on mitochondrial respiration. State 3 respiration titration with carboxyatractyloside revealed that MC inhibits the adenine nucleotide translocase. Furthermore, we analyze MC channel-forming capacity in the lipid membrane bilayer. Our results confirmed the crucial role of Δψ and showed a dependence on Ca(2+) for MC to have an effect on mitochondria. Our data also indicated that outer and inner mitochondrial membranes contribute differently to this effect, involving the presence of Δψ (the inner membrane) and VDAC (the outer membrane). Data from three isosmotic media indicate that MC does not increase the permeability of the inner membrane to protons or potassium. In addition, by using mitoplasts and ruthenium red, we showed that Ca(2+) uptake is not involved in the MC effect, suggesting involvement of VDAC in the MC interaction with the outer membrane. Our data contribute to unravel the mechanisms behind the mitochondria-targeted activity of the cytoprotective drug MC.

Methadone induces necrotic-like cell death in SH-SY5Y cells by an impairment of mitochondrial ATP synthesis.

Methadone is a widely used therapeutic opioid in narcotic addiction and neuropathic pain syndromes. Oncologists regularly use methadone as a long-lasting analgesic. Recently it has also been proposed as a promising agent in leukemia therapy, especially when conventional therapies are not effective. Nevertheless, numerous reports indicate a negative impact on human cognition with chronic exposure to opiates. Thus, clarification of methadone toxicity is required. In SH-SY5Y cells we found that high concentrations of methadone were required to induce cell death. Methadone-induced cell death seems to be related to necrotic processes rather than typical apoptosis. Cell cultures challenged with methadone presented alterations in mitochondrial outer membrane permeability. A mechanism that involves Bax translocation to the mitochondria was observed, accompanied with cytochrome c release. Furthermore, no participation of known protein regulators of apoptosis such as Bcl-X(L) and p53 was observed. Interestingly, methadone-induced cell death took place by a caspases-independent pathway; perhaps due to its ability to induce a drastic depletion in cellular ATP levels. Therefore, we studied the effect of methadone on isolated rat liver mitochondria. We observed that methadone caused mitochondrial uncoupling, coinciding with the ionophoric properties of methadone, but did not cause swelling of the organelles. Overall, the effects observed for cells in the presence of supratherapeutic doses of methadone may result from a "bioenergetic crisis." A decreased level of cellular energy may predispose cells to necrotic-like cell death.

Methadone induces CAD degradation and AIF-mediated necrotic-like cell death in neuroblastoma cells.

Methadone (d,l-methadone hydrochloride) is a full-opioid agonist, originally developed as a substitution for heroin or other opiates abusers. Nowadays methadone is also being applied as long-lasting analgesics in cancer, and it is proposed as a promising agent for leukemia therapy. Previously, we have demonstrated that high concentrations of methadone (0.5mM) induced necrotic-like cell death in SH-SY5Y cells. The pathway involved is caspase-independent but involves impairment of mitochondrial ATP synthesis and mitochondrial cytochrome c release. However, the downstream mitochondrial pathways remained unclear. Here, we studied the participation of apoptosis inducing factor (AIF) in methadone-induced cell death. Methadone resulted in a translocation of AIF from mitochondria to the nucleus. Translocation was inhibited by cyclosporine A, but not by lack of Bax protein. Therefore the effect seems mediated by the formation of the mitochondrial transition pore, but is apparently independent of Bax. Furthermore, methadone-treated SH-SY5Y nuclei show characteristics that are typical for stage I nuclear condensation. Methadone did not induce degradation of DNA into oligonucleosomal fragments or into high molecular weight DNA fragments. Absence of DNA fragmentation coincided with a considerable decrease in the levels of the caspase-actived endonuclase DNase and its chaperone-inhibitor ICAD. In conclusion, our results provide mechanistic insights into the molecular mechanisms that underlie methadone-induced cell death. This knowledge may prove useful to develop novel strategies to prevent toxic side-effects of methadone thereby sustaining its use as therapeutical agent against tumors.

Mitochondrial biology in Alzheimer's disease pathogenesis.

Despite the increasing knowledge of Alzheimer's disease (AD) management with novel pharmacologic agents, most of them are only transiently fixing symptomatic pathology. Currently there is rapid growth in the field of neuroprotective pharmacology and increasing focus on the involvement of mitochondria in this devastating disease. This review is directed at understanding the role of mitochondria-mediated pathways in AD and integrating basic biology of the mitochondria with knowledge of possible pharmacologic targets for AD treatment in an attempt to elucidate novel mitochondria-driven therapeutic interventions useful to both clinical and basic research.

A bibliometric evaluation of the top 100 cited natalizumab articles.

Natalizumab is being used in recurrent multiple sclerosis despite its history of market withdrawal due to lethal cases. We have carried out a bibliometric analysis of this drug from 1999 to February 2020 in order to assess the real impact of the use natalizumab with the goal to identify the key articles that sustain the current knowledge on the therapeutic possibilities of this compound. We have extracted from the Web of Science the top 100 most cited records (T100) and tabulated data on the journal, authors, publication year, number of citations, countries and institutions of publication, T100-records, citation density and citations per record of the works. The 100 most cited articles were selected from a total of 32,507 citations out of 2817 publications with an h-number of 74, 11.54 citations/publication, and a density of 1544.79 citations/year. Citations ranged from 63 of the paper placed in the 100th position (T100) to 1940 of the paper in the first position (T1). T2 was cited 888 times, and the difference in the number of citations between T1 and T2 was higher than that between T2 and T10. T1, T2 and T3 are clinical trials. When articles are arranged by institution and nationality having more than 10 T100 articles, biotechnology company Biogen and the USA, respectively, lead the ranking, but we also find that 8 out of 10 are academic European institutions. A co-authorship analysis reveals an intense collaborative activity between countries and institutions. We conclude that the clinical and academic communities have shown a sustained interest in natalizumab for the therapy of recurrent multiple sclerosis over the last 20 years.

6-Hydroxydopamine activates the mitochondrial apoptosis pathway through p38 MAPK-mediated, p53-independent activation of Bax and PUMA.

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used toxin to study Parkinson's disease. In previous work, we have demonstrated that 6-OHDA increases mitochondrial membrane permeability leading to cytochrome c release, but the precise mechanisms involved in this process remain unknown. Herein we studied the mechanism of increased mitochondrial permeability of SH-SY5Y neuroblastoma cells in response to 6-OHDA. Cytochrome c release induced by 6-OHDA occurred, in both SH-SY5Y cells and primary cultures, in the absence of mitochondrial swelling or a decrease in mitochondrial calcein fluorescence, suggesting little involvement of the mitochondrial permeability transition pore in this process. In contrast, 6-OHDA-induced cell death was associated with a significant translocation of the pro-apoptotic Bax protein from the cytosol to mitochondria and with a significant induction of the BH3-only protein PUMA. Experiments in mouse embryonic fibroblasts deficient in Bax or PUMA demonstrated a role for both proteins in 6-OHDA-induced apoptosis. Although 6-OHDA elevated both total and nuclear p53 protein levels, activation of p53 was not essential for subsequent cell death. In contrast, we found that p38 mitogen-activated protein kinase (MAPK) was activated early during 6-OHDA-induced apoptosis, and that treatment with the p38 MAPK inhibitor SKF86002 potently inhibited PUMA induction, green fluorescent protein-Bax redistribution and apoptosis in response to 6-OHDA. These data demonstrate a critical involvement of p38 MAPK, PUMA, and Bax in 6-OHDA-induced apoptosis.

6-Hydroxydopamine (6-OHDA) induces Drp1-dependent mitochondrial fragmentation in SH-SY5Y cells.

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used neurotoxin to study Parkinson's disease. Herein we studied the potential effects of 6-OHDA on mitochondrial morphology in SH-SY5Y neuroblastoma cells. By immunofluorescence and time-lapse fluorescence microscopy we demonstrated that 6-OHDA induced profound mitochondrial fragmentation in SH-SY5Y cells, an event that was similar to mitochondrial fission induced by overexpression of Fis1p, a membrane adaptor for the dynamin-related protein 1 (DLP1/Drp1). 6-OHDA failed to induce any changes in peroxisome morphology. Biochemical experiments revealed that 6-OHDA-induced mitochondrial fragmentation is an early event preceding the collapse of the mitochondrial membrane potential and cytochrome c release in SH-SY5Y cells. Silencing of DLP1/Drp1, which is involved in mitochondrial and peroxisomal fission, prevented 6-OHDA-induced fragmentation of mitochondria. Furthermore, in cells silenced for Drp1, 6-OHDA-induced cell death was reduced, indicating that a block in mitochondrial fission protects SH-SY5Y cells against 6-OHDA toxicity. Experiments in mouse embryonic fibroblasts deficient in Bax or p53 revealed that both proteins are not essential for 6-OHDA-induced mitochondrial fragmentation. Our data demonstrate for the first time an involvement of mitochondrial fragmentation and Drp1 function in 6-OHDA-induced apoptosis.

Guidelines on experimental methods to assess mitochondrial dysfunction in cellular models of neurodegenerative diseases.

Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the progressive degeneration and death of neurons. Mitochondrial dysfunction has been implicated in most neurodegenerative diseases, but in many instances it is unclear whether such dysfunction is a cause or an effect of the underlying pathology, and whether it represents a viable therapeutic target. It is therefore imperative to utilise and optimise cellular models and experimental techniques appropriate to determine the contribution of mitochondrial dysfunction to neurodegenerative disease phenotypes. In this consensus article, we collate details on and discuss pitfalls of existing experimental approaches to assess mitochondrial function in in vitro cellular models of neurodegenerative diseases, including specific protocols for the measurement of oxygen consumption rate in primary neuron cultures, and single-neuron, time-lapse fluorescence imaging of the mitochondrial membrane potential and mitochondrial NAD(P)H. As part of the Cellular Bioenergetics of Neurodegenerative Diseases (CeBioND) consortium ( www.cebiond.org ), we are performing cross-disease analyses to identify common and distinct molecular mechanisms involved in mitochondrial bioenergetic dysfunction in cellular models of Alzheimer's, Parkinson's, and Huntington's diseases. Here we provide detailed guidelines and protocols as standardised across the five collaborating laboratories of the CeBioND consortium, with additional contributions from other experts in the field.

Rotenone Induces the Formation of 4-Hydroxynonenal Aggresomes. Role of ROS-Mediated Tubulin Hyperacetylation and Autophagic Flux Disruption.

Oxidative stress causes cellular damage by (i) altering protein stability, (ii) impairing organelle function, or (iii) triggering the formation of 4-HNE protein aggregates. The catabolic process known as autophagy is an antioxidant cellular response aimed to counteract these stressful conditions. Therefore, autophagy might act as a cytoprotective response by removing impaired organelles and aggregated proteins. In the present study, we sought to understand the role of autophagy in the clearance of 4-HNE protein aggregates in ARPE-19 cells under rotenone exposure. Rotenone induced an overproduction of reactive oxygen species (ROS), which led to an accumulation of 4-HNE inclusions, and an increase in the number of autophagosomes. The latter resulted from a disturbed autophagic flux rather than an activation of the autophagic synthesis pathway. In compliance with this, rotenone treatment induced an increase in LC3-II while upstream autophagy markers such as Beclin- 1, Vsp34 or Atg5-Atg12, were decreased. Rotenone reduced the autophagosome-to-lysosome fusion step by increasing tubulin acetylation levels through a ROS-mediated pathway. Proof of this is the finding that the free radical scavenger, N-acetylcysteine, restored autophagy flux and reduced rotenone-induced tubulin hyperacetylation. Indeed, this dysfunctional autophagic response exacerbates cell death triggered by rotenone, since 3-methyladenine, an autophagy inhibitor, reduced cell mortality, while rapamycin, an inductor of autophagy, caused opposite effects. In summary, we shed new light on the mechanisms involved in the autophagic responses disrupted by oxidative stress, which take place in neurodegenerative diseases such as Huntington or Parkinson diseases, and age-related macular degeneration.

Malonate induces cell death via mitochondrial potential collapse and delayed swelling through an ROS-dependent pathway.

1. Herein we study the effects of the mitochondrial complex II inhibitor malonate on its primary target, the mitochondrion. 2. Malonate induces mitochondrial potential collapse, mitochondrial swelling, cytochrome c (Cyt c) release and depletes glutathione (GSH) and nicotinamide adenine dinucleotide coenzyme (NAD(P)H) stores in brain-isolated mitochondria. 3. Although, mitochondrial potential collapse was almost immediate after malonate addition, mitochondrial swelling was not evident before 15 min of drug presence. This latter effect was blocked by cyclosporin A (CSA), Ruthenium Red (RR), magnesium, catalase, GSH and vitamin E. 4. Malonate added to SH-SY5Y cell cultures produced a marked loss of cell viability together with the release of Cyt c and depletion of GSH and NAD(P)H concentrations. All these effects were not apparent in SH-SY5Y cells overexpressing Bcl-xL. 5. When GSH concentrations were lowered with buthionine sulphoximine, cytoprotection afforded by Bcl-xL overexpression was not evident anymore. 6. Taken together, all these data suggest that malonate causes a rapid mitochondrial potential collapse and reactive oxygen species production that overwhelms mitochondrial antioxidant capacity and leads to mitochondrial swelling. Further permeability transition pore opening and the subsequent release of proapoptotic factors such as Cyt c could therefore be, at least in part, responsible for malonate-induced toxicity.

Reactive oxygen species induce swelling and cytochrome c release but not transmembrane depolarization in isolated rat brain mitochondria.

1 In this study, we have used isolated brain mitochondria to investigate the effects of superoxide anions (O(2)(-)) on mitochondrial parameters related to apoptosis, such as swelling, potential, enzymatic activity, NAD(P)H, cytochrome c release, and caspase activity. 2 Addition of the reactive oxygen species (ROS) generator KO(2) produced brain mitochondrial swelling, which was blocked by cyclosporin A (CSA), and which was Ca(2+) independent. 3 Calcium induced mitochondrial swelling only at high concentrations and in the presence of succinate. This correlated with the increase in O(2)(-) production detected with hydroethidine in mitochondrial preparations exposed to Ca(2+) and the fact that ROS were required for Ca(2+)-induced mitochondrial swelling. 4 Superoxide anions, but not Ca(2+), decreased citrate synthase and dehydrogenase enzymatic activities and dropped total mitochondrial NAD(P)H levels. 5 Calcium, but not O(2)(-), triggered a rapid loss of mitochondrial potential. Calcium-induced Deltapsi(m) dissipation was inhibited by Ruthenium Red, but not by CSA. 6 Calcium- and superoxide-induced mitochondrial swelling released cytochrome c and increased caspase activity from isolated mitochondria in a CS A-sensitive manner. 7 In summary, superoxide potently triggers mitochondrial swelling and the release of proteins involved in activation of postmitochondrial apoptotic pathways in the absence of mitochondrial depolarization.

Superoxide anions mediate veratridine-induced cytochrome c release and caspase activity in bovine chromaffin cells.

1. Mitochondrial mechanisms involved in veratridine-induced chromaffin cell death have been explored. 2. Exposure to veratridine (30 micro M, 1 h) produces cytochrome c release to the cytoplasm that seems to be mediated by superoxide anions and that is blocked by cyclosporin A (10 micro M), MnTBAP (10 nM), catalase (100 IU ml(-1)) and vitamin E (50 micro M). 3. Following veratridine treatment, there is an increase in caspase-like activity, blocked by vitamin E (50 micro M) and the mitochondrial permeability transition pore blocker cyclosporin A (10 micro M). 4. Superoxide anions open the mitochondrial permeability transition pore in isolated mitochondria, an effect that is blocked by vitamin E (50 micro M) and cyclosporin A (10 micro M), but not by the Ca2+ uniporter blocker ruthenium red (5 micro M). 5. These results strongly suggest that under the stress situation caused by veratridine, superoxide anions become important regulators of mitochondrial function in chromaffin cells. 6. Exposure of isolated bovine chromaffin mitochondria to Ca2+ results in mitochondrial swelling. This effect was prevented by ruthenium red (5 micro M) and cyclosporin A (10 micro M), while it was not modified by vitamin E (50 micro M). 7. Veratridine (30 micro M, 1 h) markedly decreased total glutathione and GSH content in bovine chromaffin cells. 8. In conclusion, superoxide anions seem to mediate veratridine-induced cytochrome c release, decrease in total glutathione, caspase activation and cell death in bovine chromaffin cells.

3-Nitropropionic acid induces autophagy by forming mitochondrial permeability transition pores rather than activating the mitochondrial fission pathway.

BACKGROUND AND PURPOSE: Huntington's disease is a neurodegenerative process associated with mitochondrial alterations. Inhibitors of the electron-transport channel complex II, such as 3-nitropropionic acid (3NP), are used to study the molecular and cellular pathways involved in this disease. We studied the effect of 3NP on mitochondrial morphology and its involvement in macrophagy. EXPERIMENTAL APPROACH: Pharmacological and biochemical methods were used to characterize the effects of 3NP on autophagy and mitochondrial morphology. SH-SY5Y cells were transfected with GFP-LC3, GFP-Drp1 or GFP-Bax to ascertain their role and intracellular localization after 3NP treatment using confocal microscopy. KEY RESULTS: Untreated SH-SY5Y cells presented a long, tubular and filamentous net of mitochondria. After 3NP (5 mM) treatment, mitochondria became shorter and rounder. 3NP induced formation of mitochondrial permeability transition pores, both in cell cultures and in isolated liver mitochondria, and this process was inhibited by cyclosporin A. Participation of the mitochondrial fission pathway was excluded because 3NP did not induce translocation of the dynamin-related protein 1 (Drp1) to the mitochondria. The Drp1 inhibitor Mdivi-1 did not affect the observed changes in mitochondrial morphology. Finally, scavengers of reactive oxygen species failed to prevent mitochondrial alterations, while cyclosporin A, but not Mdivi-1, prevented the generation of ROS. CONCLUSIONS AND IMPLICATIONS: There was a direct correlation between formation of mitochondrial permeability transition pores and autophagy induced by 3NP treatment. Activation of autophagy preceded the apoptotic process and was mediated, at least partly, by formation of reactive oxygen species and mitochondrial permeability transition pores.

The senescence-accelerated mouse prone-8 (SAM-P8) oxidative stress is associated with upregulation of renal NADPH oxidase system.

Herein, we investigate whether the NADPH oxidase might be playing a key role in the degree of oxidative stress in the senescence-accelerated mouse prone-8 (SAM-P8). To this end, the activity and expression of the NADPH oxidase, the ratio of glutathione and glutathione disulfides (GSH/GSSG), and the levels of malonyl dialdehyde (MDA) and nitrotyrosine (NT) were determined in renal tissue from SAM-P8 mice at the age of 1 and 6 months. The senescence-accelerated-resistant mouse (SAM-R1) was used as control. At the age of 1 month, NADPH oxidase activity and Nox2 protein expression were higher in SAM-P8 than in SAM-R1 mice. However, we found no differences in the GSH/GSSG ratio, MDA, NT, and Nox4 levels between both groups of animals. At the age of 6 months, SAM-R1 mice in comparison to SAM-P8 mice showed an increase in NADPH oxidase activity, which is associated with higher levels of NT and increased Nox4 and Nox2 expression levels. Furthermore, we found oxidative stress hallmarks including depletion in GSH/GSSG ratio and increase in MDA levels in the kidney of SAM-P8 mice. Finally, NADPH oxidase activity positively correlated with Nox2 expression in all the animals (r = 0.382, P < 0.05). Taken together, our data allow us to suggest that an increase in NADPH oxidase activity might be an early hallmark to predict future oxidative stress in renal tissue during the aging process that takes place in SAM-P8 mice.