Maria E. Solesio

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

Parkinsons 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.

Overexpression of CB2 cannabinoid receptors results in neuroprotection against behavioral and neurochemical alterations induced by intracaudate administration of 6-hydroxydopamine

The role of CB2 cannabinoid receptors in the behavioral and neurochemical changes induced by intracaudate administration of 6-hydroxydopamine (6-OHDA) was evaluated. 6-OHDA (12 μg/4 μL) or its vehicle was injected in the caudate-putamen (CPu) of mice overexpressing the CB2 cannabinoid receptor (CB2xP) and wild type (WT) mice. Motor impairment, emotional behavior, and cognitive alterations were evaluated. Tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), and ionized calcium-binding adapter molecule 1 (Iba-1) were measured by immunocytochemistry in the CPu and/or substantia nigra (SN) of CB2xP mice and WT mice. Oxidative/nitrosative and neuroinflammatory parameters were also measured in the CPu and cortex of 6-OHDA-treated and sham-treated mice. 6-OHDA-treated CB2xP mice presented significantly less motor deterioration than 6-OHDA-treated WT mice. Immunocytochemical analysis of tyrosine hydroxylase in the SN and CPu revealed significantly fewer lesions in CB2xP mice than in WT mice. GFAP and Iba-1 immunostaining revealed less astrocyte and microglia recruitment to the treated area of the CPu in CB2xP mice. Malonyldialdehyde (MDA) concentrations were lower in the striatum and cerebral cortex of sham-treated CB2xP mice than in sham-treated WT mice. The administration of 6-OHDA increased MDA levels in both WT mice and CB2xP mice; it increased the oxidized (GSSG)/reduced (GSH) glutathione ratio in the striatum in WT mice alone compared with matched sham-treated controls. The results revealed that overexpression of CB2 cannabinoid receptors decreased the extent of motor impairment and dopaminergic neuronal loss, reduced the recruitment of astrocytes and microglia to the lesion, and decreased the level of various oxidative parameters. These results suggest that CB2 receptors offer neuroprotection against dopaminergic injury.

3‐Nitropropionic acid induces autophagy by forming mitochondrial permeability transition pores rather than activatiing the mitochondrial fission pathway

BACKGROUND AND PURPOSE Huntingtons 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.

Characterization of mitophagy in the 6-hydoxydopamine Parkinson’s disease model

In the present study, the activation of autophagy and its interaction with the mitochondrial fission machinery was investigated in an experimental model of Parkinsons disease. The addition of 50µM 6-hydroxydopamine (6-OHDA) to the dopaminergic cell line SH-SY5Y profoundly stimulated formation of autophagosomes within 12h. Under these conditions, mitochondrial fission was also activated in a sustained manner, but this occurred at earlier time points (after 3h). Upon 6-OHDA treatment, dynamin-related protein 1 (Drp1) transiently translocated to mitochondria, with increased levels of mitochondrial Drp1 being observed after 3 and 9h. Pharmacological inhibition of Drp1, through treatment with the mitochondrial-division inhibitor-1 (mdivi-1), resulted in the abrogation of mitochondrial fission and in a decrease of the number of autophagic cells. In addition, 6-OHDA failed to induce the expression of the proapoptotic protein Bax in total cellular extracts although it did induce its migration to mitochondria. In our model, Bax migrated later than Drp1. However, Drp1 inhibition did not block Bax migration. These results show that reactive oxygen species but not quinone derivates act as mediators of autophagy at an early stage of the process. 6-OHDA induces hydrogen peroxide production, which was placed upstream of mitochondrial fission, given that mdivi-1 did not abrogate this increase. Furthermore, the 6-OHDA-induced activation of autophagy was also suppressed by addition of the free radical scavengers TEMPOL and MnTBAP. This effect could be reproduced by the addition of hydrogen peroxide, but not with aged 6-OHDA. To our knowledge, this is the first detailed study highlighting the various mediators that are implicated in mitochondrial alterations and autophagy of cells in response to 6-OHDA.

Lactacystin requires reactive oxygen species and Bax redistribution to induce mitochondria-mediated cell death.

Background and purpose:  The proteasome inhibitor model of Parkinsons disease (PD) appears to reproduce many of the important behavioural, imaging, pathological and biochemical features of the human disease. However, the mechanisms involved in the lactacystin‐induced, mitochondria‐mediated apoptotic pathway remain poorly defined.

Mitochondrial dynamics and mitophagy in the 6-hydroxydopamine preclinical model of Parkinson's disease.

We discuss the participation of mitochondrial dynamics and autophagy in the 6-hydroxidopamine-induced Parkinsons disease model. The regulation of dynamic mitochondrial processes such as fusion, fission, and mitophagy has been shown to be an important mechanism controlling cellular fate. An imbalance in mitochondrial dynamics may contribute to both familial and sporadic neurodegenerative diseases including Parkinsons disease. With special attention we address the role of second messengers as the role of reactive oxygen species and the mitochondria as the headquarters of cell death. The role of molecular signaling pathways, for instance, the participation of Dynamin-related protein 1(Drp1), will also be addressed. Furthermore evidence demonstrates the therapeutic potential of small-molecule inhibitors of mitochondrial division in Parkinsons disease. For instance, pharmacological inhibition of Drp1, through treatment with the mitochondrial division inhibitor-1, results in the abrogation of mitochondrial fission and in a decrease of the number of autophagic cells. Deciphering the signaling cascades that underlie mitophagy triggered by 6-OHDA, as well as the mechanisms that determine the selectivity of this response, will help to better understand this process and may have impact on human treatment strategies of Parkinsons disease.

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.

Pharmacological Characterization of the Mechanisms Involved in Delayed Calcium Deregulation in SH-SY5Y Cells Challenged with Methadone

Previously, we have shown that SH-SY5Y cells exposed to high concentrations of methadone died due to a necrotic-like cell death mechanism related to delayed calcium deregulation (DCD). In this study, we show that, in terms of their Ca2+ responses to 0.5 mM methadone, SH-SY5Y cells can be pooled into four different groups. In a broad pharmacological survey, the relevance of different Ca2+-related mechanisms on methadone-induced DCD was investigated including extracellular calcium, L-type Ca2+ channels, μ-opioid receptor, mitochondrial inner membrane potential, mitochondrial ATP synthesis, mitochondrial Ca2+/2Na+-exchanger, reactive oxygen species, and mitochondrial permeability transition. Only those compounds targeting mitochondria such as oligomycin, FCCP, CGP 37157, and cyclosporine A were able to amend methadone-induced Ca2+ dyshomeostasis suggesting that methadone induces DCD by modulating the ability of mitochondria to handle Ca2+. Consistently, mitochondria became dramatically shorter and rounder in the presence of methadone. Furthermore, analysis of oxygen uptake by isolated rat liver mitochondria suggested that methadone affected mitochondrial Ca2+ uptake in a respiratory substrate-dependent way. We conclude that methadone causes failure of intracellular Ca2+ homeostasis, and this effect is associated with morphological and functional changes of mitochondria. Likely, this mechanism contributes to degenerative side effects associated with methadone treatment.

Role of Mitochondrial Fission and Mitophagy in Parkinson’s Disease

The involvement of mitochondrial dysfunction as a causal factor of Parkinson’s disease (PD) is well established. Impaired mitochondrial function is a predominant feature of PD. Here, we discuss mitochondrial fission and mitophagy, two processes that are described in PD. In experimental models as well as in samples from PD patients, mitochondrial fission has been shown to occur as an early step during these neurodegenerative processes. In these processes a machinery of proteins actively participates, including dynamin related protein 1 and fish. The deleterious effects of oxidative stress, resulting from increased levels of reactive oxygen species, including membrane and protein damage, are well recognized as important inducers of mitochondrial fission. Here, we discuss the current knowledge and prevailing hypotheses regarding mitochondrial dysfunction in either genetic or sporadic PD. While many questions remain unanswered, the prevalence of mitochondrial fission as an early event in neurodegenerative diseases warrants further exploration of all of these areas for development of potential treatments. Understanding the nature of these events, how they are activated, and their relative contributions to PD-mediated toxicity may strengthen future studies that aim to develop therapeutic prevention and intervention.

Mitochondrial Alterations and Mitophagy in Response to 6-Hydroxydopamine

6-Hydroxydopamine (6-OHDA) is a hydroxylated analogue of dopamine that has been exploited as an experimental model to study Parkinson’s disease (PD). In this study, we highlight the participation and relevance of mitochondrial alterations and mitophagy in response to 6-OHDA. Mitochondria play important roles in life and death of eukaryotic cell processes via oxidative phosphorylation. Mitochondrial dysfunction, either functional or morphological, has been widely associated with the cytotoxic effect of 6-OHDA. PD patients present deficiencies in the oxidative phosphorylation system. Complex I activities were found to be significantly reduced in postmortem substantia nigra of PD patients. In line with this, 6-OHDA diminishes complex I and IV activities. Mitochondrial membrane permeabilization, a critical event during apoptosis that leads to the release of cytochrome c from mitochondria, is induced by 6-OHDA. We also discuss the effect of 6-OHDA on morphological mitochondrial dynamics processes. Irreversible mitochondrial swelling does not take place in this model. The pro-apoptotic protein Bax is responsible for mitochondrial cytochrome c release. 6-OHDA induced mitochondrial fragmentation by a mechanism in which the mitochondrial fission machinery is involved. Furthermore, Drp1 translocates from the cytosol to the mitochondria in the presence of 6-OHDA. Finally, we discuss the role of reactive oxygen species as second messengers in these processes.