Reactive oxygen species and p38 mitogen-activated protein kinase activate Bax to induce mitochondrial cytochrome c release and apoptosis in response to malonate.

Malonate, an inhibitor of mitochondrial complex II, is a widely used toxin to study neurodegeneration in Huntington's disease and ischemic stroke. We have shown previously that malonate increased reactive oxygen species (ROS) production in human SH-SY5Y neuroblastoma cells, leading to oxidative stress, cytochrome c release, and apoptotic cell death. Expression of a green fluorescent protein-Bax fusion protein in SH-SY5Y neuroblastoma cells demonstrated a Bax redistribution from the cytosol to mitochondria after 12 to 24 h of malonate treatment that coincided with mitochondrial potential collapse and chromatin condensation. Inhibition of Bax translocation using furosemide, as well as Bax gene deletion, afforded significant protection against malonate-induced apoptosis. Further experiments revealed that malonate induced a prominent increase in the level of activated p38 mitogen-activated protein (MAP) kinase and that treatment with the p38 MAP kinase inhibitor SKF86002 potently blocked malonate-induced Bax translocation and apoptosis. Treatment with vitamin E diminished ROS production, reduced the activation status of p38 MAP kinase, inhibited Bax translocation, and protected against malonate-induced apoptosis. Our data suggest that malonate-induced ROS production and subsequent p38 MAP kinase activation mediates the activation of the pro-apoptotic Bax protein to induce mitochondrial membrane permeabilization and neuronal apoptosis.

Pyruvate protects cerebellar granular cells from 6-hydroxydopamine-induced cytotoxicity by activating the Akt signaling pathway and increasing glutathione peroxidase expression.

Parkinson disease (PD) is the second-most common age-related neurodegenerative disease and is characterized by the selective destruction of dopaminergic neurons. Increasing evidence indicates that oxidative stress plays a crucial role in the pathogenesis of idiopathic PD. Anti-oxidant agents including catalase, manganese porphyrin and pyruvate confer cytoprotection to different cell cultures when challenged with 6-hydroxydopamine (6-OHDA). Herein we used rat cerebellar granular cell cultures to ascertain the plausible cellular pathways involved in pyruvate-induced cytoprotection against 0.1 mM 6-OHDA. Pyruvate provided cytoprotection in a concentration-dependent manner (2-10 mM). Consistent with its well-established anti-oxidant capacity, pyruvate (10 mM) prevented 6-OHDA-induced lipid peroxidation by blocking the rise in intracellular peroxides and maintaining the intracellular reduced glutathione (GSH) levels. Further experiments revealed that pyruvate increased Akt, but not extracellular signal-regulated kinase phosphorylation. Moreover, phosphatidylinositol 3-kinase (PI3K) inhibitors attenuated pyruvate-induced cytoprotection indicating that PI3K-mediated Akt activation is necessary for pyruvate to induce cytoprotection. On the other hand, pyruvate also up-regulated glutathione peroxidase mRNA levels, but not those of the anti-oxidant enzymes superoxide dismutase-1 and -2, catalase or the anti-apoptotic oncogenes Bcl-2 or Bcl-xL. In summary, our results strongly suggest that pyruvate, besides the anti-oxidant properties related to its structure, exerts cytoprotective actions by activating different anti-apoptotic routes that include gene regulation and Akt pathway activation.

Minocycline fails to protect cerebellar granular cell cultures against malonate-induced cell death.

Experimental and clinical studies support the view that the semisynthetic tetracycline minocycline exhibits neuroprotective roles in several models of neurodegenerative diseases, including ischemia, Huntington, Parkinson diseases, and amyotrophic lateral sclerosis. However, recent evidence indicates that minocycline does not always present beneficial actions. For instance, in an in vivo model of Huntington's disease, it fails to afford protection after malonate intrastriatal injection. Moreover, it reverses the neuroprotective effect of creatine in nigrostriatal dopaminergic neurons. This apparent contradiction prompted us to analyze the effect of this antibiotic on malonate-induced cell death. We show that, in rat cerebellar granular cells, the succinate dehydrogenase inhibitor malonate induces cell death in a concentration-dependent manner. By using DFCA, monochlorobimane and 10-N-nonyl-Acridin Orange to measure, respectively, H2O2-derived oxidant species and reduced forms of GSH and cardiolipin, we observed that malonate induced reactive oxygen species (ROS) production to an extent that surpasses the antioxidant defense capacity of the cells, resulting in GSH depletion and cardiolipin oxidation. The pre-treatment for 4 h with minocycline (10-100 microM) did not present cytoprotective actions. Moreover, minocycline failed to block ROS production and to abrogate malonate-induced oxidation of GSH and cardiolipin. Additional experiments revealed that minocycline was also unsuccessful to prevent the mitochondrial swelling induced by malonate. Furthermore, malonate did not induce the expression of the iNOS, caspase-3, -8, and -9 genes which have been shown to be up-regulated in several models where minocycline resulted cytoprotective. In addition, malonate-induced down-regulation of the antiapoptotic gene Bcl-2 was not prevented by minocycline, controversially the mechanism previously proposed to explain minocycline protective action. These results suggest that the minocycline protection observed in several neurodegenerative disease models is selective, since it is absent from cultured cerebellar granular cells challenged with malonate.

Activation of p53 and the pro-apoptotic p53 target gene PUMA during depolarization-induced apoptosis of chromaffin cells.

The pathogenesis of non-glutamatergic, depolarization-induced cell death is still enigmatic. Recently, we have shown that veratridine induces apoptosis in chromaffin cells, and we have demonstrated protective effects of antioxidants in this system, suggesting a role for Na+ channels and oxidative stress in depolarization-induced cell death. We examined the possible contribution of p53, a transcription factor that has a major role in determining cell fate, and the mitochondrial apoptosis pathway in veratridine-induced cell death of cultured bovine chromaffin cells. Nuclear condensation and fragmentation were detected several hours after a 60-min exposure to 30 microM veratridine. Apoptosis was associated with a transitory increase in p53 protein levels. Veratridine induced transcription of the pro-apoptotic p53 target gene PUMA, but not of bax or pig3. Using transient transfection experiments, we found that wild-type p53, but not the mutant form p53-273H, was sufficient to induce cell death in the chromaffin cells, which was caspase-9 dependent. The down-regulation of either p53, by overexpressing p53-273H, or caspase-9 activity using a dominant-negative caspase-9 mutant protected chromaffin cells against veratridine-induced toxicity. Our data demonstrate the importance of p53 and the downstream activation of the mitochondrial apoptosis pathway in depolarization-induced apoptosis.

Involvement of mitochondrial potential and calcium buffering capacity in minocycline cytoprotective actions.

Minocycline, a semisynthetic derivative of tetracycline, displays beneficial activity in neuroprotective in models including, Parkinson disease, spinal cord injury, amyotrophic lateral sclerosis, Huntington disease and stroke. The mechanisms by which minocycline inhibits apoptosis remain poorly understood. In the present report we have investigated the effects of minocycline on mitochondria, due to their crucial role in apoptotic pathways. In mitochondria isolated suspensions, minocycline failed to block superoxide-induced swelling but was effective in blocking mitochondrial swelling induced by calcium. This latter effect might be mediated through dissipation of mitochondrial transmembrane potential and blockade of mitochondrial calcium uptake. Consistently, minocycline fails to protect SH-SY5Y cell cultures against reactive oxygen species-mediated cell death, including malonate and 6-hydroxydopamine treatments, but it is effective against staurosporine-induced cytotoxicity. The effects of this antibiotic on mitochondrial respiratory chain complex were also analyzed. Minocycline did not modify complex IV activity, and only at the higher concentration tested (100 microM) inhibited complex II/III activity. Other members of the minocycline antibiotic family like tetracycline failed to induce these mitochondrial effects.

La proteína p53 en procesos neurodegenerativos en sus 25 años de historia / The role of protein p53 in neurodegenerative processes throughout the 25 years of its history

Objetivo. A lo largo de esta revisión estudiaremos la proteína p53 en los procesos neurodegenerativos, y profundizaremos en los mecanismos de regulación de sus niveles y actividad biológica. Las neuronopatologías donde esta proteína se implica, como las enfermedades de Alzheimer, Parkinson y la esclerosis lateral amiotrófica, se analizarán, y nos adentraremos en su regulación por segundos mensajeros como las especies reactivas del oxígeno y el calcio, mostrando las rutas de señalización que intervienen en los procesos apoptóticos. Desarrollo. En el año 2004 se cumplen 25 años desde que la proteína p53 se describió por primera vez. En un principio y de forma errónea a p53, se le atribuyó una función oncogénica debido a su capacidad de unión al antígeno T del virus SV40 en células transformadas. No obstante, no fue hasta el año 1989 cuando se le atribuyó su verdadera función fisiológica como proteína supresora de tumores. Este hito constituye el punto de inflexión en la corta vida de esta proteína. Conclusiones. p53 desempeña una función fundamental en los mecanismos de respuesta celular frente al daño o mutación en el genoma. p53 puede activar dos mecanismos de señalización que conducen, bien a la parada del ciclo celular o a la muerte por apoptosis de la célula, si a ésta le resulta imposible subsanar el daño en el genoma. Sus deleciones y mutaciones se correlacionan con el desarrollo de cáncer y aumentos en su forma nativa se han descrito en patologías donde los procesos apoptóticos se encuentran elevados, como son algunas enfermedades neurodegenerativas (AU)

Aims. In this review we will study the role of protein p53 in neurodegenerative processes and conduct a detailed analysis of the mechanisms responsible for regulating its levels and biological activity. We analyse the neuropathologies in which this protein is involved, such as Alzheimer’s and Parkinson’s diseases and amyotrophic lateral sclerosis, and we will also examine its regulation by second messengers such as the reactive species of oxygen and calcium, showing the signalling paths involved in the apoptotic processes. Development. The year 2004 sees the 25th anniversary of the discovery of protein p53. At first p53 was wrongly attributed with an oncogenic function due to its capacity to bind to the T antigen of the virus SV40 in transformed cells. Nevertheless, it was not until 1989 that it was attributed with its true physiological function as a tumour-suppressing protein. This milestone constitutes a turning point in the short life of this protein. Conclusions. Protein p53 plays an essential role in the mechanisms by which the cell responds to damage or mutation in the genome. It can activate two signalling mechanisms that lead either to stopping the cell cycle or to the death of the cell due to apoptosis if the cell cannot repair the damage to the genome. There is a correlation between its deletions and mutations and the development of cancer, and increases in its native form have been described in pathologies where apoptotic processes are high, as is the case of some neurodegenerative diseases (AU)

[The role of protein p53 in neurodegenerative processes throughout the 25 years of its history]. / La proteína p53 en procesos neurodegerativos en sus 25 años de historia.

INTRODUCTION: In this review we will study the role of protein p53 in neurodegenerative processes and conduct a detailed analysis of the mechanisms responsible for regulating its levels and biological activity. We analyse the neuropathologies in which this protein is involved, such as Alzheimer's and Parkinson's diseases and amyotrophic lateral sclerosis, and we will also examine its regulation by second messengers such as the reactive species of oxygen and calcium, showing the signalling paths involved in the apoptotic processes. DEVELOPMENT: The year 2004 sees the 25th anniversary of the discovery of protein p53. At first p53 was wrongly attributed with an oncogenic function due to its capacity to bind to the T antigen of the virus SV40 in transformed cells. Nevertheless, it was not until 1989 that it was attributed with its true physiological function as a tumour-suppressing protein. This milestone constitutes a turning point in the short life of this protein. Protein p53 plays a fundamental role in the mechanisms the cell uses to respond to damage or mutation in the genome. There is, therefore, a correlation between deletions or mutations in the p53 gene and the development of some kinds of cancer; additionally, increases in the protein levels of its native form have been reported in pathologies where apoptotic processes are high. CONCLUSIONS: Protein p53 plays an essential role in the mechanisms by which the cell responds to damage or mutation in the genome. It can activate two signalling mechanisms that lead either to stopping the cell cycle or to the death of the cell due to apoptosis if the cell cannot repair the damage to the genome. There is a correlation between its deletions and mutations and the development of cancer, and increases in its native form have been described in pathologies where apoptotic processes are high, as is the case of some neurodegenerative diseases.

p53: twenty five years understanding the mechanism of genome protection.

This year the p53 protein, also known as "guardian of the genome", turns twenty five years old. During this period the p53 knowledge have changed from an initial pro-oncogene activity to the tumorsupressor p53 function. p53 is activated upon stress signals, such as gamma irradiation, UV, hypoxia, virus infection, and DNA damage, leading to protection of cells by inducing target genes. The molecules activated by p53 induce cell cycle arrest, DNA repair to conserve the genome and apoptosis. The regulation of p53 functions is tightly controlled through several mechanisms including p53 transcription and translation, protein stability, post-translational modifications, and subcellular localization. In fact, mutations in p53 are the most frequent molecular alterations detected in human tumours. Furthermore, in some degenerative processes, fragmentation and oxidative damage in DNA take place, and in these situations p53 is involved. So, p53 is considered a pharmacological target, p53 overexpression induces apoptosis in cancer and its expression blockage protects cells against lethal insults.