RESUMEN
AIMS: The study of the intracellular oxido-reductive (redox) state is of extreme relevance to the dopamine (DA) neurons of the substantia nigra pars compacta. These cells possess a distinct physiology intrinsically associated with elevated reactive oxygen species production, and they selectively degenerate in Parkinson's disease under oxidative stress conditions. To test the hypothesis that these cells display a unique redox response to mild, physiologically relevant oxidative insults when compared with other neuronal populations, we sought to develop a novel method for quantitatively assessing mild variations in intracellular redox state. RESULTS: We have developed a new imaging strategy to study redox variations in single cells, which is sensitive enough to detect changes within the physiological range. We studied DA neurons' physiological redox response in biological systems of increasing complexity--from primary cultures to zebrafish larvae, to mammalian brains-and identified a redox response that is distinctive for substantia nigra pars compacta DA neurons. We studied simultaneously, and in the same cells, redox state and signaling activation and found that these phenomena are synchronized. INNOVATION: The redox histochemistry method we have developed allows for sensitive quantification of intracellular redox state in situ. As this method is compatible with traditional immunohistochemical techniques, it can be applied to diverse settings to investigate, in theory, any cell type of interest. CONCLUSION: Although the technique we have developed is of general interest, these findings provide insights into the biology of DA neurons in health and disease and may have implications for therapeutic intervention.
Asunto(s)
Mapeo Encefálico/métodos , Dopamina/fisiología , Inmunohistoquímica/métodos , Neuronas/fisiología , Análisis de la Célula Individual , Sustancia Negra/fisiología , Animales , Astrocitos/citología , Células Cultivadas , Neuronas/citología , Oxidación-Reducción , Enfermedad de Parkinson/patología , Ratas , Especies Reactivas de Oxígeno/metabolismo , Pez Cebra/fisiologíaRESUMEN
In addition to their well-established role in providing the cell with ATP, mitochondria are the source of iron-sulfur clusters (ISCs) and heme - prosthetic groups that are utilized by proteins throughout the cell in various critical processes. The post-transcriptional system that mammalian cells use to regulate intracellular iron homeostasis depends, in part, upon the synthesis of ISCs in mitochondria. Thus, proper mitochondrial function is crucial to cellular iron homeostasis. Many neurodegenerative diseases are marked by mitochondrial impairment, brain iron accumulation, and oxidative stress - pathologies that are inter-related. This review discusses the physiological role that mitochondria play in cellular iron homeostasis and, in so doing, attempts to clarify how mitochondrial dysfunction may initiate and/or contribute to iron dysregulation in the context of neurodegenerative disease. We review what is currently known about the entry of iron into mitochondria, the ways in which iron is utilized therein, and how mitochondria are integrated into the system of iron homeostasis in mammalian cells. Lastly, we turn to recent advances in our understanding of iron dysregulation in two neurodegenerative diseases (Alzheimer's disease and Parkinson's disease), and discuss the use of iron chelation as a potential therapeutic approach to neurodegenerative disease.
Asunto(s)
Hierro/metabolismo , Mitocondrias/metabolismo , Degeneración Nerviosa/patología , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Adenosina Trifosfato/metabolismo , Animales , Homeostasis , Humanos , Proteínas Reguladoras del Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Modelos Biológicos , Degeneración Nerviosa/metabolismoRESUMEN
The cannabinoid 1 (CB(1)) and cannabinoid 2 (CB(2)) receptor agonist Delta(9)-tetrahydrocannabinol (THC) has been shown to be a broad-range inhibitor of cancer in culture and in vivo, and is currently being used in a clinical trial for the treatment of glioblastoma. It has been suggested that other plant-derived cannabinoids, which do not interact efficiently with CB(1) and CB(2) receptors, can modulate the actions of Delta(9)-THC. There are conflicting reports, however, as to what extent other cannabinoids can modulate Delta(9)-THC activity, and most importantly, it is not clear whether other cannabinoid compounds can either potentiate or inhibit the actions of Delta(9)-THC. We therefore tested cannabidiol, the second most abundant plant-derived cannabinoid, in combination with Delta(9)-THC. In the U251 and SF126 glioblastoma cell lines, Delta(9)-THC and cannabidiol acted synergistically to inhibit cell proliferation. The treatment of glioblastoma cells with both compounds led to significant modulations of the cell cycle and induction of reactive oxygen species and apoptosis as well as specific modulations of extracellular signal-regulated kinase and caspase activities. These specific changes were not observed with either compound individually, indicating that the signal transduction pathways affected by the combination treatment were unique. Our results suggest that the addition of cannabidiol to Delta(9)-THC may improve the overall effectiveness of Delta(9)-THC in the treatment of glioblastoma in cancer patients.
Asunto(s)
Cannabidiol/farmacología , Dronabinol/farmacología , Glioblastoma/patología , Apoptosis/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Glioblastoma/enzimología , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Invasividad Neoplásica , Proteínas de Neoplasias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptor Cannabinoide CB2/metabolismoRESUMEN
More than 80 years after iron accumulation was initially described in the substantia nigra (SN) of Parkinson's disease (PD) patients, the mechanisms responsible for this phenomenon are still unknown. Similarly, how iron is delivered to its major recipients in the cell - mitochondria and the respiratory complexes - has yet to be elucidated. Here, we report a novel transferrin/transferrin receptor 2 (Tf/TfR2)-mediated iron transport pathway in mitochondria of SN dopamine neurons. We found that TfR2 has a previously uncharacterized mitochondrial targeting sequence that is sufficient to import the protein into these organelles. Importantly, the Tf/TfR2 pathway can deliver Tf bound iron to mitochondria and to the respiratory complex I as well. The pathway is redox-sensitive and oxidation of Tf thiols to disulfides induces release from Tf of highly reactive ferrous iron, which contributes to free radical production. In the rotenone model of PD, Tf accumulates in dopamine neurons, with much of it accumulating in the mitochondria. This is associated with iron deposition in SN, similar to what occurs in PD. In the human SN, TfR2 is also found in mitochondria of dopamine neurons, and in PD there is a dramatic increase of oxidized Tf in SN. Thus, we have discovered a novel mitochondrial iron transport system that goes awry in PD, and which may provide a new target for therapeutic intervention.
Asunto(s)
Hierro/metabolismo , Mitocondrias/fisiología , Enfermedad de Parkinson Secundaria/metabolismo , Receptores de Transferrina/metabolismo , Sustancia Negra/fisiopatología , Transferrina/metabolismo , Anciano , Animales , Dopamina/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Humanos , Macaca fascicularis , Macaca mulatta , Neuronas/fisiología , Oxidación-Reducción , Enfermedad de Parkinson/fisiopatología , Enfermedad de Parkinson Secundaria/inducido químicamente , Ratas , Ratas Endogámicas Lew , Rotenona , Transducción de SeñalRESUMEN
Invasion and metastasis of aggressive breast cancer cells is the final and fatal step during cancer progression, and is the least understood genetically. Clinically, there are still limited therapeutic interventions for aggressive and metastatic breast cancers available. Clearly, effective and nontoxic therapies are urgently required. Id-1, an inhibitor of basic helix-loop-helix transcription factors, has recently been shown to be a key regulator of the metastatic potential of breast and additional cancers. Using a mouse model, we previously determined that metastatic breast cancer cells became significantly less invasive in vitro and less metastatic in vivo when Id-1 was down-regulated by stable transduction with antisense Id-1. It is not possible at this point, however, to use antisense technology to reduce Id-1 expression in patients with metastatic breast cancer. Here, we report that cannabidiol (CBD), a cannabinoid with a low-toxicity profile, could down-regulate Id-1 expression in aggressive human breast cancer cells. The CBD concentrations effective at inhibiting Id-1 expression correlated with those used to inhibit the proliferative and invasive phenotype of breast cancer cells. CBD was able to inhibit Id-1 expression at the mRNA and protein level in a concentration-dependent fashion. These effects seemed to occur as the result of an inhibition of the Id-1 gene at the promoter level. Importantly, CBD did not inhibit invasiveness in cells that ectopically expressed Id-1. In conclusion, CBD represents the first nontoxic exogenous agent that can significantly decrease Id-1 expression in metastatic breast cancer cells leading to the down-regulation of tumor aggressiveness.