RESUMEN
OBJECTIVE: Acute pancreatitis is caused by toxins that induce acinar cell calcium overload, zymogen activation, cytokine release and cell death, yet is without specific drug therapy. Mitochondrial dysfunction has been implicated but the mechanism not established. DESIGN: We investigated the mechanism of induction and consequences of the mitochondrial permeability transition pore (MPTP) in the pancreas using cell biological methods including confocal microscopy, patch clamp technology and multiple clinically representative disease models. Effects of genetic and pharmacological inhibition of the MPTP were examined in isolated murine and human pancreatic acinar cells, and in hyperstimulation, bile acid, alcoholic and choline-deficient, ethionine-supplemented acute pancreatitis. RESULTS: MPTP opening was mediated by toxin-induced inositol trisphosphate and ryanodine receptor calcium channel release, and resulted in diminished ATP production, leading to impaired calcium clearance, defective autophagy, zymogen activation, cytokine production, phosphoglycerate mutase 5 activation and necrosis, which was prevented by intracellular ATP supplementation. When MPTP opening was inhibited genetically or pharmacologically, all biochemical, immunological and histopathological responses of acute pancreatitis in all four models were reduced or abolished. CONCLUSIONS: This work demonstrates the mechanism and consequences of MPTP opening to be fundamental to multiple forms of acute pancreatitis and validates the MPTP as a drug target for this disease.
Asunto(s)
Células Acinares , Proteínas de Transporte de Membrana Mitocondrial , Proteínas Mitocondriales/metabolismo , Páncreas , Pancreatitis Aguda Necrotizante , Fosfoproteínas Fosfatasas/metabolismo , Células Acinares/efectos de los fármacos , Células Acinares/metabolismo , Células Acinares/patología , Animales , Autofagia/efectos de los fármacos , Calcio/metabolismo , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Humanos , Fosfatos de Inositol/metabolismo , Fosfatos de Inositol/farmacología , Ratones , Mitocondrias/enzimología , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Necrosis , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Páncreas/patología , Pancreatitis Aguda Necrotizante/inducido químicamente , Pancreatitis Aguda Necrotizante/metabolismo , Pancreatitis Aguda Necrotizante/patologíaRESUMEN
Huntington's disease is a fatal human neurodegenerative disorder caused by a CAG repeat expansion in the HTT gene, which translates into a mutant huntingtin protein. A key event in the molecular pathogenesis of Huntington's disease is the proteolytic cleavage of mutant huntingtin, leading to the accumulation of toxic protein fragments. Mutant huntingtin cleavage has been linked to the overactivation of proteases due to mitochondrial dysfunction and calcium derangements. Here, we investigated the therapeutic potential of olesoxime, a mitochondria-targeting, neuroprotective compound, in the BACHD rat model of Huntington's disease. BACHD rats were treated with olesoxime via the food for 12 months. In vivo analysis covered motor impairments, cognitive deficits, mood disturbances and brain atrophy. Ex vivo analyses addressed olesoxime's effect on mutant huntingtin aggregation and cleavage, as well as brain mitochondria function. Olesoxime improved cognitive and psychiatric phenotypes, and ameliorated cortical thinning in the BACHD rat. The treatment reduced cerebral mutant huntingtin aggregates and nuclear accumulation. Further analysis revealed a cortex-specific overactivation of calpain in untreated BACHD rats. Treated BACHD rats instead showed significantly reduced levels of mutant huntingtin fragments due to the suppression of calpain-mediated cleavage. In addition, olesoxime reduced the amount of mutant huntingtin fragments associated with mitochondria, restored a respiration deficit, and enhanced the expression of fusion and outer-membrane transport proteins. In conclusion, we discovered the calpain proteolytic system, a key player in Huntington's disease and other neurodegenerative disorders, as a target of olesoxime. Our findings suggest that olesoxime exerts its beneficial effects by improving mitochondrial function, which results in reduced calpain activation. The observed alleviation of behavioural and neuropathological phenotypes encourages further investigations on the use of olesoxime as a therapeutic for Huntington's disease.
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Calpaína/metabolismo , Colestenonas/farmacología , Colestenonas/uso terapéutico , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/metabolismo , Proteínas Mutantes/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Proteolisis/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Calpaína/antagonistas & inhibidores , Colestenonas/sangre , Colestenonas/metabolismo , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Proteína Huntingtina , Enfermedad de Huntington/enzimología , Enfermedad de Huntington/genética , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ratas , Ratas TransgénicasRESUMEN
AIM: The MITOCARE study evaluated the efficacy and safety of TRO40303 for the reduction of reperfusion injury in patients undergoing revascularization for ST-elevation myocardial infarction (STEMI). METHODS: Patients presenting with STEMI within 6 h of the onset of pain randomly received TRO40303 (n = 83) or placebo (n = 80) via i.v. bolus injection prior to balloon inflation during primary percutaneous coronary intervention in a double-blind manner. The primary endpoint was infarct size expressed as area under the curve (AUC) for creatine kinase (CK) and for troponin I (TnI) over 3 days. Secondary endpoints included measures of infarct size using cardiac magnetic resonance (CMR) and safety outcomes. RESULTS: The median pain-to-balloon time was 180 min for both groups, and the median (mean) door-to-balloon time was 60 (38) min for all sites. Infarct size, as measured by CK and TnI AUCs at 3 days, was not significantly different between treatment groups. There were no significant differences in the CMR-assessed myocardial salvage index (1-infarct size/myocardium at risk) (mean 52 vs. 58% with placebo, P = 0.1000), mean CMR-assessed infarct size (21.9 g vs. 20.0 g, or 17 vs. 15% of LV-mass) or left ventricular ejection fraction (LVEF) (46 vs. 48%), or in the mean 30-day echocardiographic LVEF (51.5 vs. 52.2%) between TRO40303 and placebo. A greater number of adjudicated safety events occurred in the TRO40303 group for unexplained reasons. CONCLUSION: This study in STEMI patients treated with contemporary mechanical revascularization principles did not show any effect of TRO40303 in limiting reperfusion injury of the ischaemic myocardium.
Asunto(s)
Angioplastia de Balón/métodos , Cardiotónicos/administración & dosificación , Infarto del Miocardio/terapia , Daño por Reperfusión Miocárdica/prevención & control , Oximas/administración & dosificación , Secoesteroides/administración & dosificación , Área Bajo la Curva , Cardiotónicos/efectos adversos , Terapia Combinada , Oclusión Coronaria/patología , Oclusión Coronaria/terapia , Método Doble Ciego , Femenino , Humanos , Angiografía por Resonancia Magnética , Masculino , Persona de Mediana Edad , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Poro de Transición de la Permeabilidad Mitocondrial , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/patología , Oximas/efectos adversos , Estudios Prospectivos , Secoesteroides/efectos adversos , Resultado del TratamientoRESUMEN
Cholesterol-oximes TRO19622 and TRO40303 target outer mitochondrial membrane proteins and have beneficial effects in preclinical models of neurodegenerative diseases leading to their advancement to clinical trials. Dopaminergic neurons degenerate in Parkinson's disease (PD) and are prone to oxidative stress and mitochondrial dysfunction. In order to provide insights into the neuroprotective potential of TRO19622 and TRO40303 for dopaminergic neurons in vivo, we assessed their effects on gene expression in laser captured nigrostriatal dopaminergic neurons of wildtype mice and of mice that over-express alpha-synuclein, a protein involved in both familial and sporadic forms of PD (Thy1-aSyn mice). Young mice were fed the drugs in food pellets or a control diet from 1 to 4months of age, approximately 10months before the appearance of striatal dopamine loss in this model. Unbiased weighted gene co-expression network analysis (WGCNA) of transcriptional changes revealed effects of cholesterol oximes on transcripts related to mitochondria, cytoprotection and anti-oxidant response in wild-type and transgenic mice, including increased transcription of stress defense (e.g. Prdx1, Prdx2, Glrx2, Hspa9, Pink1, Drp1, Trak1) and dopamine-related (Th, Ddc, Gch1, Dat, Vmat2, Drd2, Chnr6a) genes. Even at this young age transgenic mice showed alterations in transcripts implicated in mitochondrial function and oxidative stress (e.g. Bcl-2, Bax, Casp3, Nos2), and both drugs normalized about 20% of these alterations. Young Thy1-aSyn mice exhibit motor deficits that differ from parkinsonism and are established before the onset of treatment; these deficits were not improved by cholesterol oximes. However, high doses of TRO40303 improved olfaction and produced the same effects as dopamine agonists on a challenging beam test, specifically an increase in footslips, an observation congruent with its effects on transcripts involved in dopamine synthesis. High doses of TRO19622 increased alpha-synuclein aggregates in the substantia nigra; this effect, not seen with TRO40303 was inconsistent and may represent a protective mechanism as in other neurodegenerative diseases. Overall, the results suggest that cholesterol oximes, while not improving early effects of alpha-synuclein overexpression on motor behavior or pathology, may ameliorate the function and resilience of dopaminergic neurons in vivo and support further studies of neuroprotection in models with dopaminergic cell loss.
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Encéfalo/efectos de los fármacos , Colestenonas/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Oximas/farmacología , Secoesteroides/farmacología , alfa-Sinucleína/metabolismo , Animales , Encéfalo/metabolismo , Colestenonas/farmacocinética , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Agonistas de Dopamina/farmacología , Neuronas Dopaminérgicas/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones Transgénicos , Trastornos del Movimiento/tratamiento farmacológico , Trastornos del Movimiento/metabolismo , Fármacos Neuroprotectores/farmacocinética , Oximas/farmacocinética , Trastornos Parkinsonianos/tratamiento farmacológico , Trastornos Parkinsonianos/metabolismo , ARN Mensajero/metabolismo , Secoesteroides/farmacocinética , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Transcriptoma/efectos de los fármacos , alfa-Sinucleína/genéticaRESUMEN
OBJECTIVE: Multiple sclerosis is a neurodegenerative disease characterized by episodes of immune attack of oligodendrocytes leading to demyelination and progressive functional deficit. One therapeutic strategy to address disease progression could consist in stimulating the spontaneous regenerative process observed in some patients. Myelin regeneration requires endogenous oligodendrocyte progenitor migration and activation of the myelination program at the lesion site. In this study, we have tested the ability of olesoxime, a neuroprotective and neuroregenerative agent, to promote remyelination in the rodent central nervous system in vivo. METHODS: The effect of olesoxime on oligodendrocyte progenitor cell (OPC) differentiation and myelin synthesis was tested directly in organotypic slice cultures and OPC-neuron cocultures. Using naive animals and different mouse models of demyelination, we morphologically and functionally assessed the effect of the compound on myelination in vivo. RESULTS: Olesoxime accelerated oligodendrocyte maturation and enhanced myelination in vitro and in vivo in naive animals during development and also in the adult brain without affecting oligodendrocyte survival or proliferation. In mouse models of demyelination and remyelination, olesoxime favored the repair process, promoting myelin formation with consequent functional improvement. INTERPRETATION: Our observations support the strategy of promoting oligodendrocyte maturation and myelin synthesis to enhance myelin repair and functional recovery. We also provide proof of concept that olesoxime could be useful for the treatment of demyelinating diseases.
Asunto(s)
Colestenonas/uso terapéutico , Enfermedades Desmielinizantes/tratamiento farmacológico , Esclerosis Múltiple/tratamiento farmacológico , Vaina de Mielina/efectos de los fármacos , Oligodendroglía/efectos de los fármacos , Animales , Cuprizona/toxicidad , Enfermedades Desmielinizantes/inducido químicamente , Modelos Animales de Enfermedad , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Inhibidores de la Monoaminooxidasa/toxicidad , Esclerosis Múltiple/fisiopatología , Vaina de Mielina/fisiología , Oligodendroglía/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor neuron (SMN) protein leading to muscle paralysis and respiratory failure. In mouse, introducing the human SMN2 gene partially rescues Smn(-)(/)(-) embryonic lethality. However current models were either too severe or nearly unaffected precluding convenient drug testing for SMA. We report here new SMN2;Smn(-/-) lines carrying one to four copies of the human SMN2 gene. Mice carrying three SMN2 copies exhibited an intermediate phenotype with delayed appearance of motor defects and developmental breathing disorders reminiscent of those found in severe SMA patients. Although normal at birth, at 7 days of age respiratory rate was decreased and apnea frequency was increased in SMA mice in parallel with the appearance of neuromuscular junction defects in the diaphragm. With median survival of 15 days and postnatal onset of neurodegeneration, these mice could be an important tool for evaluating new therapeutics.
Asunto(s)
Atrofia Muscular Espinal/fisiopatología , Enfermedades de la Unión Neuromuscular/fisiopatología , Parálisis Respiratoria/fisiopatología , Animales , Diafragma/inervación , Diafragma/fisiopatología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Genes Letales/fisiología , Predisposición Genética a la Enfermedad/genética , Humanos , Ratones , Ratones Transgénicos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Unión Neuromuscular/genética , Unión Neuromuscular/metabolismo , Unión Neuromuscular/patología , Enfermedades de la Unión Neuromuscular/genética , Enfermedades de la Unión Neuromuscular/metabolismo , Insuficiencia Respiratoria/genética , Insuficiencia Respiratoria/metabolismo , Insuficiencia Respiratoria/fisiopatología , Parálisis Respiratoria/genética , Parálisis Respiratoria/metabolismo , Proteína 2 para la Supervivencia de la Neurona Motora/genéticaRESUMEN
3,5-Seco-4-nor-cholestan-5-one oxime-3-ol (TRO40303) is a new cardioprotective compound coming from a chemical series identified initially for neuroprotective properties. TRO40303 binds specifically to the mitochondrial translocator protein 18 kDa (TSPO) at the cholesterol site. After intravenous administration, TRO40303 tissue distribution was comparable to that of TSPO, and, in particular, the drug accumulated rapidly in the heart. In a model of 35 min of myocardial ischemia/24 h of reperfusion in rats, TRO40303 (2.5 mg/kg) reduced infarct size by 38% (p < 0.01 versus control), when administered 10 min before reperfusion, which was correlated with reduced release of apoptosis-inducing factor from mitochondria to the cytoplasm in the ischemic area at risk. Although TRO40303 had no effect on the calcium retention capacity of isolated mitochondria, unlike cyclosporine A, the drug delayed mitochondrial permeability transition pore (mPTP) opening and cell death in isolated adult rat cardiomyocytes subjected to 2 h of hypoxia followed by 2 h of reoxygenation and inhibited mPTP opening in neonatal rat cardiomyocytes treated with hydrogen peroxide. The effects of TRO40303 on mPTP in cell models of oxidative stress are correlated with a significant reduction in reactive oxygen species production and subsequent calcium overload. TRO40303 is a new mitochondrial-targeted drug and inhibits mPTP triggered by oxidative stress. Its mode of action differs from that of other mPTP inhibitors such as cyclosporine A, thus providing a new pharmacological approach to study mPTP regulation. Its efficacy in an animal model of myocardial infarctions makes TRO40303 a promising new drug for the reduction of cardiac ischemia-reperfusion injury.
Asunto(s)
Cardiotónicos/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Oximas/farmacología , Secoesteroides/farmacología , Animales , Animales Recién Nacidos , Western Blotting , Calcio/metabolismo , Cardiotónicos/metabolismo , Cardiotónicos/farmacocinética , Muerte Celular/efectos de los fármacos , Células Cultivadas , Citosol/efectos de los fármacos , Citosol/metabolismo , Peróxido de Hidrógeno/toxicidad , Inyecciones Intravenosas , Masculino , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Membranas Mitocondriales/efectos de los fármacos , Infarto del Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Oxidantes/toxicidad , Estrés Oxidativo/efectos de los fármacos , Oximas/metabolismo , Oximas/farmacocinética , Permeabilidad/efectos de los fármacos , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Secoesteroides/metabolismo , Secoesteroides/farmacocinética , Distribución TisularRESUMEN
Diabetes and cancer chemotherapies are often associated with painful neuropathy. The mechanisms underlying neuropathic pain remain poorly understood, and the current therapies have limited efficacy and are associated with dose-limiting side effects. We recently described the pharmacological characterization of cholest-4-en-3-one, oxime (TRO19622), a cholesterol-like compound, that significantly reduced axonal degeneration and accelerated recovery of motor nerve conduction in a model of peripheral neuropathy induced by crushing the sciatic nerve. These results triggered investigation of efficacy in other preclinical models of peripheral neuropathy. Here, we report evidence that daily oral administration of TRO19622, while similarly improving motor nerve conduction impaired in streptozotocin-induced diabetic rats, also reversed neuropathic pain behavior as early as the first administration. Further exploration of these acute antinociceptive effects demonstrated that TRO19622 was also able to reverse tactile allodynia in vincristine-treated rats, a model of chemotherapy-induced neuropathic pain. It is interesting to note that TRO19622 did not have analgesic activity in animal models of pain produced by formalin injection, noxious thermal or mechanical stimulation, or chronic constriction injury of the sciatic nerve, indicating that painful diabetic or chemotherapy-induced neuropathies share a common mechanism that is distinct from acute, inflammationdriven, or lesion-induced neuropathic pain. These results support the potential use of TRO19622 to treat painful diabetic and chemotherapy-induced neuropathies.
Asunto(s)
Analgésicos , Conducta Animal/efectos de los fármacos , Colestenonas , Diabetes Mellitus Experimental/complicaciones , Dolor/tratamiento farmacológico , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Analgésicos/sangre , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , Antineoplásicos Fitogénicos/efectos adversos , Colestenonas/sangre , Colestenonas/farmacología , Colestenonas/uso terapéutico , Diabetes Mellitus Experimental/fisiopatología , Neuropatías Diabéticas/tratamiento farmacológico , Neuropatías Diabéticas/etiología , Neuropatías Diabéticas/fisiopatología , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/etiología , Hiperalgesia/fisiopatología , Masculino , Conducción Nerviosa/efectos de los fármacos , Dolor/fisiopatología , Dimensión del Dolor/efectos de los fármacos , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Tiempo de Reacción/efectos de los fármacos , Estreptozocina , Vincristina/efectos adversosRESUMEN
OBJECTIVES: Mitochondrial permeability transition pore inhibition is a promising approach to treat acute pancreatitis (AP). We sought to determine (i) the effects of the mitochondrial permeability transition pore inhibitor 3,5-seco-4-nor-cholestan-5-one oxime-3-ol (TRO40303) on murine and human pancreatic acinar cell (PAC) injury induced by fatty acid ethyl esters (FAEEs) or taurolithocholic acid-3-sulfate and (ii) TRO40303 pharmacokinetics and efficacy in experimental alcoholic AP (FAEE-AP). METHODS: Changes in mitochondrial membrane potential (Δψm), cytosolic Ca ([Ca]c), and cell fate were examined in freshly isolated murine or human PACs by confocal microscopy. TRO40303 pharmacokinetics were assessed in cerulein-induced AP and therapeutic efficacy in FAEE-AP induced with palmitoleic acid and ethanol. Severity of AP was assessed by standard biomarkers and blinded histopathology. RESULTS: TRO40303 prevented loss of Δψm and necrosis induced by 100 µM palmitoleic acid ethyl ester or 500 µM taurolithocholic acid-3-sulfate in murine and human PACs. Pharmacokinetic analysis found TRO40303 accumulated in the pancreas. A single dose of 3 mg/kg TRO40303 significantly reduced serum amylase (P = 0.043), pancreatic trypsin (P = 0.018), and histopathology scores (P = 0.0058) in FAEE-AP. CONCLUSIONS: TRO40303 protects mitochondria and prevents necrotic cell death pathway activation in murine and human PACs, ameliorates the severity of FAEE-AP, and is a candidate drug for human AP.
Asunto(s)
Ésteres/farmacología , Ácidos Grasos/farmacología , Mitocondrias/efectos de los fármacos , Oximas/farmacología , Pancreatitis Alcohólica/prevención & control , Secoesteroides/farmacología , Células Acinares/efectos de los fármacos , Células Acinares/metabolismo , Enfermedad Aguda , Animales , Ceruletida , Ésteres/metabolismo , Ácidos Grasos/metabolismo , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Necrosis/prevención & control , Oximas/farmacocinética , Pancreatitis/inducido químicamente , Pancreatitis/prevención & control , Pancreatitis Alcohólica/metabolismo , Pancreatitis Alcohólica/patología , Secoesteroides/farmacocinética , Ácido Taurolitocólico/análogos & derivados , Ácido Taurolitocólico/farmacologíaRESUMEN
The expansion of a polyglutamine tract in the ubiquitously expressed huntingtin protein causes Huntington's disease (HD), a dominantly inherited neurodegenerative disease. We show that the activity of the cholesterol biosynthetic pathway is altered in HD. In particular, the transcription of key genes of the cholesterol biosynthetic pathway is severely affected in vivo in brain tissue from HD mice and in human postmortem striatal and cortical tissue; this molecular dysfunction is biologically relevant because cholesterol biosynthesis is reduced in cultured human HD cells, and total cholesterol mass is significantly decreased in the CNS of HD mice and in brain-derived ST14A cells in which the expression of mutant huntingtin has been turned on. The transcription of the genes of the cholesterol biosynthetic pathway is regulated via the activity of sterol regulatory element-binding proteins (SREBPs), and we found an approximately 50% reduction in the amount of the active nuclear form of SREBP in HD cells and mouse brain tissue. As a consequence, mutant huntingtin reduces the transactivation of an SRE-luciferase construct even under conditions of SREBP overexpression or in the presence of an exogenous N-terminal active form of SREBP. Finally, the addition of exogenous cholesterol to striatal neurons expressing mutant huntingtin prevents their death in a dose-dependent manner. We conclude that the cholesterol biosynthetic pathway is impaired in HD cells, mice, and human subjects, and that the search for HD therapies should also consider cholesterol levels as both a potential target and disease biomarker.
Asunto(s)
Colesterol/biosíntesis , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Neuronas/fisiología , Análisis de Varianza , Animales , Western Blotting/métodos , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Células Cultivadas , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Relación Dosis-Respuesta a Droga , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , Inmunohistoquímica/métodos , Ratones , Neuronas/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Transporte de Proteínas/genética , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Factores de Tiempo , Transfección/métodosRESUMEN
TRO40303 is cytoprotective compound that was shown to reduce infarct size in preclinical models of myocardial infarction. It targets mitochondria, delays mitochondrial permeability transition pore (mPTP) opening and reduces oxidative stress in cardiomyocytes submitted to ischemia/reperfusion in vitro. Because the involvement of the mitochondria and the mPTP has been demonstrated in chronic as well as acute hepatitis, we investigated the potential of TRO40303 to prevent hepatocyte injury. A first set of in vitro studies showed that TRO40303 (from 0.3 to 3 µmol/L) protected HepG2 cells and primary mouse embryonic hepatocytes (PMEH) from palmitate intoxication, a model mimicking steatohepatitis. In PMEH, TRO40303 provided similar protection against cell death due to Jo2 anti-Fas antibody intoxication. Further studies were then preformed in a mouse model of Fas-induced fulminant hepatitis induced by injecting Jo2 anti-Fas antibody. When mice received a sublethal dose of Jo2 at 125 µg/kg, TRO40303 pretreatment prevented liver enzyme elevation in plasma in parallel with a decrease in cytochrome C release from mitochondria and caspase 3 and 7 activation in hepatic tissue. When higher, lethal doses of Jo2 were administered, TRO40303 (10 and 30 mg/kg) significantly reduced mortality by 65-90% when administered intraperitoneally (i.p.) 1 h before Jo2 injection, a time when TRO40303 plasma concentrations reached their peak. TRO40303 (30 mg/kg, i.p.) was also able to reduce mortality by 30-50% when administered 1 h postlethal Jo2 intoxication. These results suggest that TRO40303 could be a promising new therapy for the treatment or prevention of hepatitis.
RESUMEN
The mode of protection against cardiac reperfusion injury by mild hypothermia and TRO40303 was investigated in various experimental models and compared to MitoQ in vitro. In isolated cardiomyocytes subjected to hypoxia/reoxygenation, TRO40303, MitoQ and mild hypothermia delayed mPTP opening, inhibited generation of mitochondrial superoxide anions at reoxygenation and improved cell survival. Mild hypothermia, but not MitoQ and TRO40303, provided protection in a metabolic starvation model in H9c2 cells and preserved respiratory function in isolated rat heart mitochondria submitted to anoxia/reoxygenation. In the Langendorff-perfused rat heart, only mild hypothermia provided protection of hemodynamic function and reduced infarct size following ischemia/reperfusion. In biopsies from the left ventricle of pigs subjected to in vivo occlusion/reperfusion, TRO40303 specifically preserved respiratory functions in the peri-infarct zone whereas mild hypothermia preserved both the ischemic core area and the peri-infarct zones. Additionally in this pig model, only hypothermia reduced infarct size. We conclude that mild hypothermia provided protection in all models by reducing the detrimental effects of ischemia, and when initiated before occlusion, reduced subsequent reperfusion damage leading to a smaller infarct. By contrast, although TRO40303 provided similar protection to MitoQ in vitro and offered specific protection against some aspects of reperfusion injury in vivo, this was insufficient to reduce infarct size.
Asunto(s)
Cardiotónicos/uso terapéutico , Hipotermia Inducida/métodos , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Oximas/uso terapéutico , Secoesteroides/uso terapéutico , Animales , Células Cultivadas , Femenino , Masculino , Daño por Reperfusión Miocárdica/terapia , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , PorcinosRESUMEN
Huntington disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the huntingtin gene (HTT). One prominent target of the mutant huntingtin protein (mhtt) is the mitochondrion, affecting its morphology, distribution, and function. Thus, mitochondria have been suggested as potential therapeutic targets for the treatment of HD. Olesoxime, a cholesterol-like compound, promotes motor neuron survival and neurite outgrowth in vitro, and its effects are presumed to occur via a direct interaction with mitochondrial membranes (MMs). We examined the properties of MMs isolated from cell and animal models of HD as well as the effects of olesoxime on MM fluidity and cholesterol levels. MMs isolated from brains of aged Hdh Q111/Q111 knock-in mice showed a significant decrease in 1,6-diphenyl-hexatriene (DPH) anisotropy, which is inversely correlated with membrane fluidity. Similar increases in MM fluidity were observed in striatal STHdh Q111/Q111 cells as well as in MMs isolated from brains of BACHD transgenic rats. Treatment of STHdh cells with olesoxime decreased the fluidity of isolated MMs. Decreased membrane fluidity was also measured in olesoxime-treated MMs isolated from brains of HD knock-in mice. In both models, treatment with olesoxime restored HD-specific changes in MMs. Accordingly, olesoxime significantly counteracted the mhtt-induced increase in MM fluidity of MMs isolated from brains of BACHD rats after 12 months of treatment in vivo, possibly by enhancing MM cholesterol levels. Thus, olesoxime may represent a novel pharmacological tool to treat mitochondrial dysfunction in HD.
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Encéfalo/metabolismo , Colestenonas/farmacología , Enfermedad de Huntington/metabolismo , Fluidez de la Membrana/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Animales , Encéfalo/efectos de los fármacos , Línea Celular , Colestenonas/uso terapéutico , Modelos Animales de Enfermedad , Enfermedad de Huntington/tratamiento farmacológico , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , RatasRESUMEN
Loss of Parkin, encoded by PARK2 gene, is a major cause of autosomal recessive Parkinson's disease. In Drosophila and mammalian cell models Parkin has been shown in to play a role in various processes essential to maintenance of mitochondrial quality, including mitochondrial dynamics, biogenesis and degradation. However, the relevance of altered mitochondrial quality control mechanisms to neuronal survival in vivo is still under debate. We addressed this issue in the brain of PARK2-/- mice using an integrated mitochondrial evaluation, including analysis of respiration by polarography or by fluorescence, respiratory complexes activity by spectrophotometric assays, mitochondrial membrane potential by rhodamine 123 fluorescence, mitochondrial DNA content by real time PCR, and oxidative stress by total glutathione measurement, proteasome activity, SOD2 expression and proteins oxidative damage. Respiration rates were lowered in PARK2-/- brain with high resolution but not standard respirometry. This defect was specific to the striatum, where it was prominent in neurons but less severe in astrocytes. It was present in primary embryonic cells and did not worsen in vivo from 9 to 24 months of age. It was not associated with any respiratory complex defect, including complex I. Mitochondrial inner membrane potential in PARK2-/- mice was similar to that of wild-type mice but showed increased sensitivity to uncoupling with ageing in striatum. The presence of oxidative stress was suggested in the striatum by increased mitochondrial glutathione content and oxidative adducts but normal proteasome activity showed efficient compensation. SOD2 expression was increased only in the striatum of PARK2-/- mice at 24 months of age. Altogether our results show a tissue-specific mitochondrial defect, present early in life of PARK2-/- mice, mildly affecting respiration, without prominent impact on mitochondrial membrane potential, whose underlying mechanisms remain to be elucidated, as complex I defect and prominent oxidative damage were ruled out.
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Cuerpo Estriado/embriología , Mitocondrias/fisiología , Superóxido Dismutasa/metabolismo , Ubiquitina-Proteína Ligasas/deficiencia , Animales , Respiración de la Célula , Cuerpo Estriado/metabolismo , Potencial de la Membrana Mitocondrial , Ratones , Especificidad de Órganos , Estrés Oxidativo , Superóxido Dismutasa/genéticaRESUMEN
Contractile dysfunction and subsequent development of cardiomyopathies are well known limiting factors in the treatment of cancer with doxorubicin and have been linked to mitochondrial dysfunction. Here, using adult isolated paced cardiomyocytes, we have demonstrated that ligands of translocator protein (TSPO) 4'-chlorodiazepam and TRO40303 prevented the doxorubicin-induced alterations in contractility and improved cardiomyocyte viability. This cardioprotective effect was closely associated with both a potent reduction in reactive oxygen species production and inhibition of mitochondrial permeability transition pore opening. Thus, preventive administration of TSPO ligands may represent a novel pharmacological strategy to protect the heart during doxorubicin treatment.
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Proteínas Portadoras/metabolismo , Muerte Celular/efectos de los fármacos , Doxorrubicina/farmacología , Mitocondrias Cardíacas/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Animales , Ligandos , Ratones , Ratones Noqueados , Miocitos Cardíacos/citología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Olesoxime is a small cholesterol-oxime promoting rat embryonic motor neurons survival in the absence of trophic factors. Because olesoxime can substitute for neurotrophic factors in many situations, and to gain further understanding of its mechanism of action, we wondered if it could prevent neuronal death induced by camptothecin (CPT) and compared its effects with those of brain-derived neurotrophic factor (BDNF). EXPERIMENTAL APPROACH: E17 rat embryonic cortical neurons were treated with olesoxime, BDNF or vehicle and intoxicated with CPT. Caspase-dependent and caspase-independent death pathways along with pro-survival pathways activation were explored. KEY RESULTS: As previously reported for BDNF, olesoxime dose-dependently delayed CPT-induced cell death. Both compounds acted downstream of p53 activation preventing cytochrome c release and caspases activation. When caspase activation was blocked, both olesoxime and BDNF provided additional neuroprotective effect, potentially through the prevention of apoptosis-inducing factor release from mitochondria. While BDNF activates both the PI3K/Akt and the ERK pathway, olesoxime induced only a late activation of the ERK pathways, which did not seem to play a major role in its neuroprotection against CPT. Rather, our results favour preserved mitochondrial membrane integrity by olesoxime. CONCLUSIONS AND IMPLICATIONS: Albeit different, olesoxime and BDNF mechanisms for neuroprotection converge to preserve mitochondrial function. These findings emphasize the importance of targeting the mitochondria in the process of neurodegeneration. Importantly olesoxime, by mimicking neurotrophin pro-survival activities without impacting PI3K/Akt and ERK signalling, may have greater therapeutic potential in many diseases where neurotrophins were considered as a therapeutic solution.
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Factor Neurotrófico Derivado del Encéfalo/farmacología , Camptotecina/toxicidad , Corteza Cerebral/embriología , Colestenonas/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neuronas/efectos de los fármacos , Animales , Butadienos/farmacología , Camptotecina/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colestenonas/administración & dosificación , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos/citología , Femenino , Regulación de la Expresión Génica , Mitocondrias/fisiología , Nitrilos/farmacología , Embarazo , Ratas , Ratas Sprague-Dawley , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
In Huntington disease (HD), there is increasing evidence for a link between mutant huntingtin expression, mitochondrial dysfunction, energetic deficits and neurodegeneration but the precise nature, causes and order of these events remain to be determined. In this work, our objective was to evaluate mitochondrial respiratory function in intact, non-permeabilized, neurons derived from a transgenic rat model for HD compared to their wild type littermates by measuring oxygen consumption rates and extracellular acidification rates. Although HD striatal neurons had similar respiratory capacity as those from their wild-type littermates when they were incubated in rich medium containing a supra-physiological glucose concentration (25 mM), pyruvate and amino acids, respiratory defects emerged when cells were incubated in media containing only a physiological cerebral level of glucose (2.5 mM). According to the concept that glucose is not the sole substrate used by the brain for neuronal energy production, we provide evidence that primary neurons can use lactate as well as pyruvate to fuel the mitochondrial respiratory chain. In contrast to glucose, we found no major deficits in HD striatal neurons' capacity to use pyruvate as a respiratory substrate compared to wild type littermates. Additionally, we used extracellular acidification rates to confirm a reduction in anaerobic glycolysis in the same cells. Interestingly, the metabolic disturbances observed in striatal neurons were not seen in primary cortical neurons, a brain region affected in later stages of HD. In conclusion, our results argue for a dysfunction in glycolysis, which might precede any defects in the respiratory chain itself, and these are early events in the onset of disease.
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Cuerpo Estriado/metabolismo , Glucosa/metabolismo , Enfermedad de Huntington/metabolismo , Neuronas/metabolismo , Animales , Respiración de la Célula , Modelos Animales de Enfermedad , Espacio Extracelular/metabolismo , Glucólisis , Ácido Láctico/metabolismo , Masculino , Ácido Pirúvico/metabolismo , Ratas , Ratas TransgénicasRESUMEN
INTRODUCTION: Spinal muscular atrophy (SMA) is an autosomal recessive disease caused by mutations in a gene that produces a protein called survival motor neuron (SMN). SMN has an important role in snRNP assembly in all cells but that may not be its only role; the reasons for SMN deficiency resulting in neuromuscular dysfunction and motor neuron degeneration remain active areas of research. Besides increasing SMN, compensating for SMN deficiencies or neuroprotection may be therapeutic options for SMA. Age of onset and the rate of disease progression are variable and therapeutic strategies should be appropriate to subtypes of SMA patients. AREAS COVERED: The article discusses SMA, their targets and where these targets can be found. Additionally, the article reviews small molecules identified as disease modifiers and how these small molecules were discovered. The article also describes and discusses emerging concepts regarding the disease mechanisms. The author compiled this review using scientific literature, patent databases, company and patient association and government websites. EXPERT OPINION: Small molecules targeting various processes implicated in SMA are reaching the clinic. These molecules and targets, although not yet validated, are providing insight into the complexity of a 'simple' genetic disease such as SMA. SMA is not a single disease and so various therapeutic strategies are needed. Biomarkers and regulatory guidelines are required to select patients for clinical trials, decide when to initiate treatment and how to develop combinations of investigational drugs.
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Olesoxime (TRO19622) is a novel mitochondrial-targeted neuroprotective compound undergoing a pivotal clinical efficacy study in Amyotrophic Lateral Sclerosis (ALS) and also in development for Spinal Muscular Atrophy (SMA). It belongs to a new family of cholesterol-oximes identified for its survival-promoting activity on purified motor neurons deprived of neurotrophic factors. Olesoxime targets proteins of the outer mitochondrial membrane, concentrates at the mitochondria and prevents permeability transition pore opening mediated by, among other things, oxidative stress. Olesoxime has been shown to exert a potent neuroprotective effect in various in vitro and in vivo models. In particular olesoxime provided significant protection in experimental animal models of motor neuron disorders and more particularly ALS. Olesoxime is orally active, crosses the blood brain barrier, and is well tolerated. Collectively, its pharmacological properties designate olesoxime as a promising drug candidate for motor neuron diseases.
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Microtubule-targeting agents (MTAs), anticancer drugs widely used in the clinic, often induce peripheral neuropathy, a main dose-limiting side effect. The mechanism for this neurotoxicity remains poorly understood and there are still no approved therapies for neuropathies triggered by MTAs. Olesoxime (cholest-4-en-3-one, oxime; TRO19622) has shown marked neuroprotective properties in animals treated with paclitaxel and vincristine. The purpose of this study was to investigate its mechanism of neuroprotection against MTA neurotoxicity by using rat and human differentiated neuronal cells. We first showed that olesoxime prevented neurite shrinkage induced by MTAs in differentiated PC-12 and SK-N-SH neuroblastoma cell lines by up to 90%. This neuroprotective effect was correlated with enhanced EB1 accumulation at microtubule plus-ends, increased growth cone microtubule growing rate (20%) and decreased microtubule attenuation duration (54%). The effects of olesoxime on EB comets were specific for differentiated neuronal cells and were not seen either in proliferating neuroblastoma cells, glioblastoma cells or primary endothelial cells. Importantly, olesoxime did not alter MTA cytotoxic properties in a wide range of MTA-sensitive tumor cells, a prerequisite for future clinical application. Finally, olesoxime also counteracted MTA inhibition of microtubule-dependent mitochondria trafficking. These results provide additional insight into the neuroprotective properties of olesoxime, highlighting a role for microtubule dynamics in preservation of neurite architecture and axoplasmic transport, which are both disturbed by MTAs. The neuron-specific protective properties of olesoxime support its further development to treat MTA-induced neuropathy.