A. Cristina Rego

Mitochondrial Dysfunction and Reactive Oxygen Species in Excitotoxicity and Apoptosis: Implications for the Pathogenesis of Neurodegenerative Diseases*

In recent years we have witnessed a major interest in the study of the role of mitochondria, not only as ATP producers through oxidative phosphorylation but also as regulators of intracellular Ca2+ homeostasis and endogenous producers of reactive oxygen species (ROS). Interestingly, the mitochondria have been also implicated as central executioners of cell death. Increased mitochondrial Ca2+ overload as a result of excitotoxicity has been associated with the generation of superoxide and may induce the release of proapoptotic mitochondrial proteins, proceeding through DNA fragmentation/condensation and culminating in cell demise by apoptosis and/or necrosis. In addition, these processes have been implicated in the pathogenesis of many neurodegenerative diseases, which share several features of cell death: selective brain areas undergo neurodegeneration, involving mitochondrial dysfunction (mitochondrial complexes are affected), loss of intracellular Ca2+ homeostasis, excitotoxicity, and the extracellular or intracellular accumulation of insoluble protein aggregates in the brain.

An Integrative View of the Role of Oxidative Stress, Mitochondria and Insulin in Alzheimer's Disease

The processes underlying the pathogenesis of Alzheimers disease involve several factors including impaired glucose/energy metabolism, mitochondrial dysfunction, oxidative stress and altered insulin-signaling pathways. This review is mainly devoted to discuss evidence supporting the notion that mitochondrial dysfunction and oxidative stress are interconnected and intimately associated with the development and progression of Alzheimers disease. Furthermore, the review explores the role of insulin signaling in the pathophysiology of the disease. Indeed, several studies have begun to find links between insulin and mechanisms with clear pathogenic implications for this disorder. Understanding the key mechanisms involved in the etiopathogenesis of Alzheimers disease may provide opportunities for the design of efficacious preventive and therapeutic strategies.

Mitochondrial-Dependent Ca2+ Handling in Huntington's Disease Striatal Cells: Effect of Histone Deacetylase Inhibitors

Evidence suggests that neuronal dysfunction in Huntingtons disease (HD) striatum involves deficits in mitochondrial function and in Ca2+ handling. However, the relationship between mitochondria and Ca2+ handling has been incompletely studied in intact HD striatal cells. Treatment with histone deacetylase (HDAC) inhibitors reduces cell death in HD models, but the effects of this promising therapy on cellular function are mostly unknown. Here, we use real-time functional imaging of intracellular Ca2+ and mitochondrial membrane potential to explore the role of in situ HD mitochondria in Ca2+ handling. Immortalized striatal (STHdh) cells and striatal neurons from transgenic mice, expressing full-length mutant huntingtin (Htt), were used to model HD. We show that (1) active glycolysis in STHdh cells occludes the mitochondrial role in Ca2+ handling as well as the effects of mitochondrial inhibitors, (2) STHdh cells and striatal neurons in the absence of glycolysis are critically dependent on oxidative phosphorylation for energy-dependent Ca2+ handling, (3) expression of full-length mutant Htt is associated with deficits in mitochondrial-dependent Ca2+ handling that can be ameliorated by treatment with HDAC inhibitors (treatment with trichostatin A or sodium butyrate decreases the proportion of STHdh cells losing Ca2+ homeostasis after Ca2+-ionophore challenging, and accelerates the restoration of intracellular Ca2+ in striatal neurons challenged with NMDA), and (4) neurons with different response patterns to NMDA receptor activation exhibit different average somatic areas and are differentially affected by treatment with HDAC inhibitors, suggesting subpopulation or functional state specificity. These findings indicate that neuroprotection induced by HDAC inhibitors involves more efficient Ca2+ handling, thus improving the neuronal ability to cope with excitotoxic stimuli.

Mitochondrial dysfunction in Huntington's disease : the bioenergetics of isolated and in situ mitochondria from transgenic mice

Mitochondrial dysfunction is believed to participate in Huntington’s disease (HD) pathogenesis. Here we compare the bioenergetic behavior of forebrain mitochondria isolated from different transgenic HD mice (R6/2, YAC128 and Hdh150 knock‐in) and wild‐type littermates with the first determination of in situ respiratory parameters in intact HD striatal neurons. We assess the Ca2+‐loading capacity of isolated mitochondria by steady Ca2+‐infusion. Mitochondria from R6/2 mice (12–13 weeks) and 12 months YAC128, but not homozygous or heterozygous Hdh150 knock‐in mice (15–17 weeks), exhibit increased Ca2+‐loading capacity when compared with respective wild‐type littermates. In situ mitochondria in intact striatal neurons show high respiratory control. Moreover, moderate expression of full‐length mutant huntingtin (in Hdh150 knock‐in heterozygotes) does not significantly impair mitochondrial respiration in unstimulated neurons. However, when challenged with energy‐demanding stimuli (NMDA‐receptor activation in pyruvate‐based media to accentuate the mitochondria role in Ca2+‐handling), Hdh150 neurons are more vulnerable to Ca2+‐deregulation than neurons from their wild‐type littermates. These results stress the importance of assessing HD mitochondrial function in the cellular context.

Effect of amyloid β-peptide on permeability transition pore: A comparative study

A potentially central factor in neurodegeneration is the permeability transition pore (PTP). Because of the tissue‐specific differences in pore properties, we directly compared isolated brain and liver mitochondria responses to the neurotoxic Aβ peptides. For this purpose, the following parameters were examined: mitochondrial membrane potential (ΔΨm), respiration, swelling, ultrastructural morphology, and content of cytochrome c. Both peptides, Aβ25–35 (50 μM) and Aβ1–40 (2 μM), had a similar toxicity, exacerbating the effects of Ca2+, although, per se, they did not induce (PTP). In liver mitochondria, Aβ led to a drop in ΔΨm and potentiated matrix swelling and disruption induced by Ca2+. In contrast, brain mitochondria, exposed to the same conditions, demonstrated a higher capacity to accumulate Ca2+ before the ΔΨm drop and a slight increase of mitochondrial swelling compared with liver mitochondria. Furthermore, mitochondrial respiratory state 3 was depressed in the presence of Aβ, whereas state 4 was unaltered, resulting in an uncoupling of respiration. In both types of mitochondria, Aβ did not affect the content of cytochrome c. The ΔΨm drop was reversed when Ca2+ was removed by EGTA or when ADP plus oligomycin was present. Pretreatment with cyclosporin A or ADP plus oligomycin prevented the deleterious effects promoted by Aβ and/or Ca2+. It can be concluded that brain and liver mitochondria show a different susceptibility to the deleterious effect of Aβ peptide, brain mitochondria being more resistant to the potentiation by Aβ of Ca2+‐induced PTP.

Mitochondrial dysfunction and caspase activation in rat cortical neurons treated with cocaine or amphetamine.

Drug abuse is associated with brain dysfunction and neurodegeneration, and various recreational drugs induce apoptotic cell death. This study examined the role of the mitochondrial apoptotic pathway in psychostimulant-induced neuronal dysfunction. Using primary neuronal cultures, we observed that amphetamine (IC50=1.40 mM) was more potent than cocaine (IC50=4.30 mM) in inducing cell toxicity. Apoptotic cell death was further evaluated using cocaine and amphetamine concentrations that moderately decreased cell reduction capacity but did not affect plasma membrane integrity. Compared to cocaine, amphetamine highly decreased the mitochondrial membrane potential, as determined using the fluorescent probe rhodamine-123, whereas both drugs decreased mitochondrial cytochrome c. In contrast to amphetamine, cocaine cytotoxicity was partly mediated through effects on the electron transport chain, since cocaine toxicity was ameliorated in mitochondrial DNA-depleted cells lacking mitochondrially encoded electron transport chain subunits. Cocaine and amphetamine induced activation of caspases-2, -3 and -9 but did not affect activity of caspases-6 or -8. In addition, amphetamine, but not cocaine, was associated with the appearance of evident nuclear apoptotic morphology. These events were not accompanied by differences in the release of the apoptosis-inducing factor (AIF) from mitochondria. Our results demonstrate that although both amphetamine and cocaine activate the mitochondrial apoptotic pathway in cortical neurons, amphetamine is more likely to promote apoptosis.

Insulin Restores Metabolic Function in Cultured Cortical Neurons Subjected to Oxidative Stress

We previously demonstrated that insulin has a neuroprotective role against oxidative stress, a deleterious condition associated with diabetes, ischemia, and age-related neurodegenerative diseases. In this study, we investigated the effect of insulin on neuronal glucose uptake and metabolism after oxidative stress in rat primary cortical neurons. On oxidative stress, insulin stimulates neuronal glucose uptake and subsequent metabolism into pyruvate, restoring intracellular ATP and phosphocreatine. Insulin also increases intracellular and decreases extracellular adenosine, counteracting the effect of oxidative stress. Insulin effects are apparently mediated by phosphatidylinositol 3-K and extracellular signal–regulated kinase signaling pathways. Extracellular adenosine under oxidative stress is largely inhibited after blockade of ecto-5′-nucleotidase, suggesting that extracellular adenosine results preferentially from ATP release and catabolism. Moreover, insulin appears to interfere with the ATP release induced by oxidative stress, regulating extracellular adenosine levels. In conclusion, insulin neuroprotection against oxidative stress–mediated damage involves 1) stimulation of glucose uptake and metabolism, increasing energy levels and intracellular adenosine and, ultimately, uric acid formation and 2) a decrease in extracellular adenosine, which may reduce the facilitatory activity of adenosine receptors.

The R6 lines of transgenic mice: A model for screening new therapies for Huntington's disease

Huntingtons disease (HD) is a hereditary neurodegenerative disorder caused by an expanded CAG repeat in the HD gene that results in cortical and striatal degeneration, and mutant huntingtin aggregation. Current treatments are unsatisfactory. R6 transgenic mice replicate many features of the human condition, show early onset of symptoms and fast disease progression, being one of the most used models for therapy screening. Here we review the therapies that have been tested in these mice: environmental enrichment, inhibition of histone deacetylation and methylation, inhibition of misfolding and oligomerization, transglutaminase inhibition, rescue of metabolic impairment, amelioration of the diabetic phenotype, use of antioxidants, inhibition of excitotoxicity, caspase inhibition, transplantation, genetic manipulations, and restoration of neurogenesis. Although many of these treatments were beneficial in R6 mice, they may not be as effective in HD patients, and thus the search for a combination of therapies that will rescue the human condition continues.

Street heroin induces mitochondrial dysfunction and apoptosis in rat cortical neurons

Cortical function has been suggested to be highly compromised by repeated heroin self‐administration. We have previously shown that street heroin induces apoptosis in neuronal‐like PC12 cells. Thus, we analysed the apoptotic pathways involved in street heroin neurotoxicity using primary cultures of rat cortical neurons. Our street heroin sample was shown to be mainly composed by heroin, 6‐monoacetylmorphine and morphine. Exposure of cortical neurons to street heroin induced a slight decrease in metabolic viability, without loss of neuronal integrity. Early activation of caspases involved in the mitochondrial apoptotic pathway was observed, culminating in caspase 3 activation, Poly‐ADP Ribose Polymerase (PARP) cleavage and DNA fragmentation. Apoptotic morphology was completely prevented by the non‐selective caspase inhibitor z‐VAD‐fmk, indicating an important role for caspases in neurodegeneration induced by street heroin. Ionotropic glutamate receptors, opioid receptors and oxidative stress were not involved in caspase 3 activation. Interestingly, street heroin cytotoxicity was shown to be independent of a functional mitochondrial respiratory chain, as determined using NT‐2 rho0 cells. Nonetheless, in street heroin‐treated cortical neurons, cytochrome c was released, accompanied by a decrease in mitochondrial potential and Bcl‐2/Bax. Pure heroin hydrochloride similarly decreased metabolic viability but only slightly activated caspase 3. Altogether, our data suggest an important role for mitochondria in mediating street heroin neurotoxic effects.

FK506 prevents mitochondrial-dependent apoptotic cell death induced by 3-nitropropionic acid in rat primary cortical cultures

The mitochondrial toxin 3-nitropropionic acid (3-NP) has been largely used to study neurodegenerative disorders in which bioenergetic defects are implicated. In the present study, we analyzed the molecular pathways involved in FK506 neuroprotection against cell death induced by 3-NP, using cultured cortical neurons. 3-NP induced cytochrome c release and increased caspases -2, -3, -8, and -9-like activities, although, calpain activity was not significantly affected. FK506 decreased cytochrome c release and caspase-3-like activity induced by 3-NP, without changing the activities of other caspases. FK-506 also decreased the number of apoptotic neurons, determined by Hoechst. Under these conditions, FK506 alone significantly reduced calcineurin activity by about 50%. Our results also showed a decrease in mitochondrial Bax and an increase in mitochondrial Bcl-2 levels upon exposure to FK506 and 3-NP. However, no significant changes occurred in total Bcl-2 and Bax levels. Altogether, the results suggest that FK506 neuroprotection against 3-NP-induced apoptosis is associated with the redistribution of Bcl-2 and Bax in the mitochondrial membrane.