RESUMEN
Parkinson's disease (PD) is an incurable neurodegenerative disorder caused by the selective loss of dopaminergic neurons in the substantia nigra pars compacta. Current therapies are only symptomatic and are not able to stop or delay its progression. In order to search for new and more effective therapies, our group carried out a high-throughput screening assay, identifying several candidate compounds that are able to improve locomotor ability in DJ-1ß mutant flies (a Drosophila model of familial PD) and reduce oxidative stress (OS)-induced lethality in DJ-1-deficient SH-SY5Y human cells. One of them was vincamine (VIN), a natural alkaloid obtained from the leaves of Vinca minor. Our results showed that VIN is able to suppress PD-related phenotypes in both Drosophila and human cell PD models. Specifically, VIN reduced OS levels in PD model flies. Besides, VIN diminished OS-induced lethality by decreasing apoptosis, increased mitochondrial viability, and reduced OS levels in DJ-1-deficient human cells. In addition, our results show that VIN might be exerting its beneficial role, at least partially, by the inhibition of voltage-gated sodium channels. Therefore, we propose that these channels might be a promising target in the search for new compounds to treat PD and that VIN represents a potential therapeutic treatment for the disease.
Asunto(s)
Proteínas de Drosophila , Neuroblastoma , Enfermedad de Parkinson , Vincamina , Animales , Humanos , Suplementos Dietéticos , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas del Tejido Nervioso/genética , Estrés Oxidativo , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/genética , Proteína Desglicasa DJ-1/genética , Proteína Desglicasa DJ-1/farmacología , Proteína Desglicasa DJ-1/uso terapéutico , Vincamina/farmacología , Vincamina/uso terapéuticoRESUMEN
Single-cell protein therapeutics is expected to promote our in-depth understanding of how a specific protein with a therapeutic dosage treats the cell without population averaging. However, it has not yet been tackled by current single-cell nanotools. We address this challenge by the use of a double-barrel nanopipette, in which one lumen was used for electroosmotic cytosolic protein delivery and the other was customized for ionic evaluation of the consequence. Upon injection of protein DJ-1 through the delivery lumen, upregulation of the antioxidant protein could protect neural PC-12 cells against oxidative stress from phorbol myristate acetate exposure, as deduced by targeting of the cytosolic hydrogen peroxide by the detecting lumen. The nanotool developed in this study for single-cell protein therapeutics provides a perspective for future single-cell therapeutics involving different therapeutic modalities, such as peptides, enzymes and nucleic acids.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Proteína Desglicasa DJ-1 , Iones , Péptidos , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Sistema de Administración de Fármacos con Nanopartículas , Proteína Desglicasa DJ-1/farmacología , Proteína Desglicasa DJ-1/uso terapéutico , Estrés Oxidativo , Acetato de TetradecanoilforbolRESUMEN
The pathophysiological process of cerebral apoplexy is complex, and there are currently no specific drugs for this condition. The study of effective drug targets has become a hot topic in neuroscience. Currently, adeno-associated viruses (AAVs) and polypeptides are commonly used in drug research. DJ-1 has been widely considered a neuroprotective target in recent times, but the mechanism of its neuroprotective effects is unclear. In this study, we simulated ischemic injury by establishing a middle cerebral artery occlusion reperfusion (MCAO/R) model to compare the protective effect of DJ-1 overexpression induced by DJ-1 AAV and ND-13 on cerebral ischemia-reperfusion (I/R) injury. We found that DJ-1 overexpression and ND-13 significantly reduced the neurological function scores and infarct volume and alleviated pathological damage to brain tissue. In addition, Western blotting, ELISA and immunofluorescence labeling revealed that DJ-1 overexpression and ND-13 increased the expression of the anti-inflammatory cytokines IL-10 and IL-4, and decreased the levels of the pro-inflammatory cytokines IL-1ß and TNF-α. In summary, our study shows that DJ-1 overexpression and ND-13 can regulate the expression of inflammatory factors and alleviate cerebral I/R injury. Thus, DJ-1 is a possible drug target for cerebral I/R injury.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Fragmentos de Péptidos/uso terapéutico , Proteína Desglicasa DJ-1/metabolismo , Proteína Desglicasa DJ-1/uso terapéutico , Daño por Reperfusión/tratamiento farmacológico , Secuencia de Aminoácidos , Animales , Encéfalo/efectos de los fármacos , Encéfalo/patología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Citocinas/metabolismo , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/patología , Inflamación/metabolismo , Inflamación/patología , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patologíaRESUMEN
DJ-1 is a redox-sensitive chaperone with reported antioxidant and anti-inflammatory properties in the kidney. The 20 amino acid (aa) peptide ND-13 consists of 13 highly conserved aas from the DJ-1 sequence and a TAT-derived 7 aa sequence that helps in cell penetration. This study aimed to determine if ND-13 treatment prevents the renal damage and inflammation associated with unilateral ureter obstruction (UUO). Male C57Bl/6 and DJ-1-/- mice underwent UUO and were treated with ND-13 or vehicle for 14 days. ND-13 attenuated the renal expression of fibrotic markers TGF-ß and collagen1a1 (Col1a1) and inflammatory markers TNF-α and IL-6 in C57Bl/6 mice. DJ-1-/- mice treated with ND-13 presented similar decreased expression of TNF-α, IL-6 and TGF-ß. However, in contrast to C57Bl/6 mice, ND-13 failed to prevent renal fibrosis or to ameliorate the expression of Col1a1 in this genotype. Further, UUO led to elevated urinary levels of the proximal tubular injury marker neutrophil gelatinase-associated lipocalin (NGAL) in DJ-1-/- mice, which were blunted by ND-13. Our results suggest that ND-13 protects against UUO-induced renal injury, inflammation and fibrosis. These are all crucial mechanisms in the pathogenesis of kidney injury. Thus, ND-13 may be a new therapeutic approach to prevent renal diseases.
Asunto(s)
Antiinflamatorios/uso terapéutico , Antioxidantes/uso terapéutico , Inflamación/tratamiento farmacológico , Fragmentos de Péptidos/uso terapéutico , Sustancias Protectoras/uso terapéutico , Proteína Desglicasa DJ-1/uso terapéutico , Obstrucción Ureteral/tratamiento farmacológico , Animales , Biomarcadores/metabolismo , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Interleucina-6/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Ischemia/reperfusion injury is a tissue injury occurring post-reperfusion of tissues with pre-existing ischemia. A good blood supply to tissues aids in the survival of ischemic tissue, however, due to prolonged ischemia the levels of ATP decrease and pH declines leading to acidosis. Reduced ATP leads to an increase in the AMP/ATP ratio, causing cessation of intracellular calcium transport, hence calcium overload and cell death. In this study, we demonstrate the synergistic and antagonistic effect of DJ1 and microR-214 (miR-214) in rescuing myoblast C2C12 cells after ischemia/reperfusion in an in vitro model. Both DJ1 and miR-214 were cloned into a hypoxic inducible expression cassette and transfected into the C2C12 cells. We showed that DJ1 and miR-214 have synergistic effects in reducing intracellular lactate dehydrogenase and intracellular transient calcium levels after reoxygenation compared to control cells, in addition to reducing cell death via necrosis. Western blotting revealed a decrease in autophagosome formation in LC3II/I ratio and an increase in AKT expression in cells transfected with DJ1 and miR-214. Using quantitative real-time PCR, we demonstrated that DJ1 and miR-214 significantly reduced the expression of pro-apoptotic factors and autophagy compared to control. The results indicated DJ1 is an endogenous oxidative stress molecule and miR-214 is a potent inhibitor of the sodium calcium exchanger channel. DJ1 had the greatest effect to inhibiting mitochondrial cell death pathways by possibly acting as a modulator of autophagy. Additionally, we have concluded that miR-214 has an inhibitory effect on extrinsic cell death pathways such as necrosis and autophagy.
Asunto(s)
Hipoxia de la Célula , MicroARNs/metabolismo , Mioblastos/metabolismo , Proteína Desglicasa DJ-1/metabolismo , Daño por Reperfusión/metabolismo , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Calcio/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , MicroARNs/uso terapéutico , Mitocondrias/metabolismo , Necrosis/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Proteína Desglicasa DJ-1/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Intercambiador de Sodio-Calcio/antagonistas & inhibidoresRESUMEN
Stroke is a leading cause of death worldwide and inflicts serious long-term damage and disability. The vasoconstrictor Endothelin-1, presenting long-term neurological deficits associated with excitotoxicity and oxidative stress is being increasingly used to induce focal ischemic injury as a model of stroke. A DJ-1 based peptide named ND-13 was shown to protect against glutamate toxicity, neurotoxic insults and oxidative stress in various animal models. Here we focus on the benefits of treatment with ND-13 on the functional outcome of focal ischemic injury. Wild type C57BL/6 mice treated with ND-13, after ischemic induction in this model, showed significant improvement in motor function, including improved body balance and motor coordination, and decreased motor asymmetry. We found that DJ-1 knockout mice are more sensitive to Endothelin-1 ischemic insult than wild type mice, contributing thereby additional evidence to the widely reported relevance of DJ-1 in neuroprotection. Furthermore, treatment of DJ-1 knockout mice with ND-13, following Endothelin-1 induced ischemia, resulted in significant improvement in motor functions, suggesting that ND-13 provides compensation for DJ-1 deficits. These preliminary results demonstrate a possible basis for clinical application of the ND-13 peptide to enhance neuroprotection in stroke patients.