RESUMEN
Iron is essential for retinal metabolism, but an excess of ferrous iron causes oxidative stress. In glaucomatous eyes, retinal ganglion cell (RGC) death has been associated with dysregulation of iron homeostasis. Transferrin (TF) is an endogenous iron transporter that controls ocular iron levels. Intraocular administration of TF is neuroprotective in various models of retinal degeneration, preventing iron overload and reducing iron-induced oxidative stress. Herein, we assessed the protective effects of TF on RGC survival, using ex vivo rat retinal explants exposed to iron, NMDA-induced excitotoxicity, or CoCl2-induced hypoxia, and an in vivo rat model of ocular hypertension (OHT). TF significantly preserved RGCs against FeSO4-induced toxicity, NMDA-induced excitotoxicity, and CoCl2-induced hypoxia. TF protected RGCs from apoptosis, ferroptosis, and necrosis. In OHT rats, TF reduced RGC loss by about 70% compared to vehicle-treated animals and preserved about 47% of the axons. Finally, increased iron staining was shown in the retina of a glaucoma patient's eye as compared to non-glaucomatous eyes. These results indicate that TF can interfere with different cell-death mechanisms involved in glaucoma pathogenesis and demonstrate the ability of TF to protect RGCs exposed to elevated IOP. Altogether, these results suggest that TF is a promising treatment against glaucoma neuropathy.
Asunto(s)
Glaucoma , Fármacos Neuroprotectores , Hipertensión Ocular , Animales , Ratas , Modelos Animales de Enfermedad , Glaucoma/metabolismo , Hipoxia , Presión Intraocular , Hierro/metabolismo , N-Metilaspartato , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Hipertensión Ocular/metabolismo , Transferrina/farmacologíaRESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
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
Non-infectious uveitis is a heterogenous group of potentially blinding ocular autoimmune diseases that may represent a manifestation of a systemic condition or may affect the eyes only. A systemically administered anti-TNF has recently been approved for the treatment of non-infectious uveitis, broadening the therapeutic arsenal available to control intraocular inflammation and reduce uveitis complications that can lead to vision loss. When uveitis affects only the eyes, a local anti-TNF-α administration strategy could optimize the ocular therapeutic effect and reduce undesirable systemic side-effects. A new ocular method of non-viral gene therapy, currently in development, may broaden the indications for ocular anti-TNF-α agents, not only for uveitis but also for other diseases in which TNF-α-mediated neuro-inflammation has been demonstrated.
TITLE: Les anti-TNF-α pour le traitement des uvéites non infectieuses. ABSTRACT: Les molécules anti-TNF-α administrés par voie générale ont été approuvés récemment pour le traitement des uvéites non inflammatoires, élargissant l'arsenal thérapeutique dans le traitement de ces pathologies responsables de cécité évitable si l'inflammation est contrôlée. Quand seul l'Åil est atteint, des stratégies d'administration locale permettraient d'optimiser les effets intraoculaires des molécules anti-TNF-α et d'en réduire les effets indésirables. Une nouvelle méthode de thérapie génique non virale, actuellement en développement, pourrait élargir les indications des molécules anti-TNF-α oculaires, non seulement pour les uvéites, mais également pour d'autres maladies dans lesquelles une neuro-inflammation impliquant le TNF-α a été démontrée.
Asunto(s)
Terapia Genética , Factor de Necrosis Tumoral alfa/inmunología , Uveítis/terapia , Trastornos de la Visión/prevención & control , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/uso terapéutico , Enfermedades Autoinmunes/terapia , Terapia Genética/métodos , Terapia Genética/tendencias , Humanos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
Dysregulation of iron metabolism is observed in animal models of retinitis pigmentosa (RP) and in patients with age-related macular degeneration (AMD), possibly contributing to oxidative damage of the retina. Transferrin (TF), an endogenous iron chelator, was proposed as a therapeutic candidate. Here, the efficacy of TF non-viral gene therapy based on the electrotransfection of pEYS611, a plasmid encoding human TF, into the ciliary muscle was evaluated in several rat models of retinal degeneration. pEYS611 administration allowed for the sustained intraocular production of TF for at least 3 and 6 months in rats and rabbits, respectively. In the photo-oxidative damage model, pEYS611 protected both retinal structure and function more efficiently than carnosic acid, a natural antioxidant, reduced microglial infiltration in the outer retina and preserved the integrity of the outer retinal barrier. pEYS611 also protected photoreceptors from N-methyl-N-nitrosourea-induced apoptosis. Finally, pEYS611 delayed structural and functional degeneration in the RCS rat model of RP while malondialdehyde (MDA) ocular content, a biomarker of oxidative stress, was decreased. The neuroprotective benefits of TF non-viral gene delivery in retinal degenerative disease models further validates iron overload as a therapeutic target and supports the continued development of pEY611 for treatment of RP and dry AMD.
RESUMEN
Ocular gene therapy has entered into clinical practice. Although viral vectors are currently the best option to replace and/or correct genes, the optimal method to deliver these treatments to the retinal pigment epithelial (RPE) cells and/or photoreceptor cells remains to be improved to increase transduction efficacy and reduce iatrogenic risks. Beyond viral-mediated gene replacement therapies, nonviral gene delivery approaches offer the promise of sustained fine-tuned expression of secreted therapeutic proteins that can be adapted to the evolving stage of the disease course and can address more common nongenetic retinal diseases, such as age-related macular degeneration (AMD). Here, we review current gene therapy strategies for ocular diseases, with a focus on clinical stage products.
Asunto(s)
Vectores Genéticos/genética , Degeneración Macular/genética , Degeneración Macular/terapia , Animales , Ojo/fisiopatología , Terapia Genética/métodos , Humanos , Epitelio Pigmentado de la Retina/fisiologíaRESUMEN
Adeno-associated viruses (AAVs) are among the most efficient vectors for liver gene therapy. Results obtained in the first hemophilia clinical trials demonstrated the long-term efficacy of this approach in humans, showing efficient targeting of hepatocytes with both self-complementary (sc) and single-stranded (ss) AAV vectors. However, to support clinical development of AAV-based gene therapies, efficient and scalable production processes are needed. In an effort to translate to the clinic an approach of AAV-mediated liver gene transfer to treat Crigler-Najjar (CN) syndrome, we developed an (ss)AAV8 vector carrying the human UDP-glucuronosyltransferase family 1-member A1 (hUGT1A1) transgene under the control of a liver-specific promoter. We compared our construct with similar (sc)AAV8 vectors expressing hUGT1A1, showing comparable potency in vitro and in vivo. Conversely, (ss)AAV8-hUGT1A1 vectors showed superior yields and product homogeneity compared with their (sc) counterpart. We then focused our efforts in the scale-up of a manufacturing process of the clinical product (ss)AAV8-hUGT1A1 based on the triple transfection of HEK293 cells grown in suspension. Large-scale production of this vector had characteristics identical to those of small-scale vectors produced in adherent cells. Preclinical studies in animal models of the disease and a good laboratory practice (GLP) toxicology-biodistribution study were also conducted using large-scale preparations of vectors. These studies demonstrated long-term safety and efficacy of gene transfer with (ss)AAV8-hUGT1A1 in relevant animal models of the disease, thus supporting the clinical translation of this gene therapy approach for the treatment of CN syndrome.
RESUMEN
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
Non-infectious uveitis (NIU) is the first cause of blindness that can be cured if optimal anti-inflammatory therapy can be achieved. Systemic anti-TNF (Tumor Necrosis Factor) agents have been recently approved for NIU but no local delivery of anti-TNF is available. For sustained production of secreted therapeutic proteins into the eye, non-viral gene therapy using plasmid electrotransfer in the ciliary muscle has been proposed. In this paper, we report the development steps of pEYS606, a clinical-grade plasmid DNA, devoid of antiobiotic selection gene, encoding a fusion protein consisting of the extracellular domain of the soluble p55 TNF-α receptor linked to the human IgG1 Fc domain (hTNFR-Is/hIgG1 or Protein 6), with high affinity for human TNF-α, for non-viral gene transfer into the ocular ciliary muscle. Electrotransfer of pEYS606 in the ciliary muscle significantly reduced ocular inflammation in two well-established rat models of uveitis, the endotoxin-induced uveitis (EIU) and the experimental autoimmune uveitis (EAU). In addition, in EAU, a significant protection of photoreceptors was demonstrated after pEYS606 treatment. The improved pharmacokinetic profile of intraocularly-secreted protein as compared to direct intravitreous injection of recombinant protein allowed to demonstrate Protein 6 efficacy at very low concentrations. Based on these results, a phase I/II clinical trial is conducted [ClinicalTrials.gov Identifier: NCT03308045].
Asunto(s)
Terapia Genética/métodos , Plásmidos/uso terapéutico , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Señuelo del Factor de Necrosis Tumoral/genética , Uveítis/terapia , Animales , Cuerpo Ciliar/metabolismo , Cuerpo Ciliar/patología , Femenino , Inmunoglobulina G/genética , Masculino , Plásmidos/genética , Conejos , Ratas Endogámicas Lew , Proteínas Recombinantes de Fusión/genética , Transfección/métodos , Uveítis/genética , Uveítis/patologíaRESUMEN
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 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.
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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
Peripheral sensorineural hearing loss is a very common inner ear disorder affecting nearly 10% of the population. At present there is no cure for this disorder but gene therapy has been suggested as a potentially effective method for clinical treatment in the future. Thus we investigated the effectiveness of adenoviral (Ad) and adeno-associated viral (AAV) vectors to transduce the cochlea of guinea pigs. After direct injection into the basal turn of the cochlea, we found that both Ad and AAV vectors coding for the reporter genes lacZ or GFP readily transduced spiral ganglion cells. In addition, some transgene expression was detected in the stria vascularis after AAV-GFP injection. Gene expression persisted at least 8 weeks after viral vector injection. Present findings will help to develop future gene therapy protocols in the inner ear by using Ad and AAV coding for neurotrophins such as NT-3, BDNF, GDNF and VEGF.
Asunto(s)
Adenoviridae/genética , Cóclea/fisiología , Técnicas de Transferencia de Gen , Vectores Genéticos , Animales , Enfermedades Cocleares/terapia , Expresión Génica , Genes Reporteros , Terapia Genética/métodos , Proteínas Fluorescentes Verdes , Cobayas , Indicadores y Reactivos/metabolismo , Operón Lac , Proteínas Luminiscentes/genéticaRESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
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.
Asunto(s)
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
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.