RESUMEN
Inflammatory processes in the brain can exert important neuroprotective functions. However, in neurological and psychiatric disorders, it is often detrimental due to chronic microglial over-activation and the dysregulation of cytokines and chemokines. Growing evidence indicates the emerging yet prominent pathophysiological role of neuroinflammation in the development and progression of these disorders. Despite recent advances, there is still a pressing need for effective therapies, and targeting neuroinflammation is a promising approach. Therefore, in this study, we investigated the anti-neuroinflammatory potential of a marketed and quantified proprietary herbal extract of Ginkgo biloba leaves called EGb 761 (10-500 µg/mL) in BV2 microglial cells stimulated by LPS (10 ng/mL). Our results demonstrate significant inhibition of LPS-induced expression and release of cytokines tumor necrosis factor-α (TNF-α) and Interleukin 6 (IL-6) and chemokines C-X-C motif chemokine ligand 2 (CXCL2), CXCL10, c-c motif chemokine ligand 2 (CCL2) and CCL3 in BV2 microglial cells. The observed effects are possibly mediated by the mitogen-activated protein kinases (MAPK), p38 MAPK and ERK1/2, as well as the protein kinase C (PKC) and the nuclear factor (NF)-κB signaling cascades. The findings of this in vitro study highlight the anti-inflammatory properties of EGb 761 and its therapeutic potential, making it an emerging candidate for the treatment of neuroinflammatory diseases and warranting further research in pre-clinical and clinical settings.
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Antiinflamatorios , Ginkgo biloba , Lipopolisacáridos , Microglía , Extractos Vegetales , Ginkgo biloba/química , Microglía/efectos de los fármacos , Microglía/metabolismo , Extractos Vegetales/farmacología , Animales , Ratones , Antiinflamatorios/farmacología , Línea Celular , Citocinas/metabolismo , FN-kappa B/metabolismo , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Extracto de GinkgoRESUMEN
Tinnitus is defined as the perception of sound in the absence of external acoustic stimuli. Frequent comorbidities or associated factors are depression, anxiety, concentration problems, insomnia, resignation, helplessness, headache, bruxism, or social isolation, just to name a few. Although many therapeutic approaches have already been tested with varying success, there still is no cure available for tinnitus. The search for an effective treatment has been hampered by the fact that the mechanisms of tinnitus development are still not fully understood, although several models are available and discussed in this review. Our review will give a brief overview about preclinical models, presenting the heterogeneity of tinnitus sub-types depending on the different inner ear and brain structures involved in tinnitus etiology and pathogenesis. Based on these models we introduce the different target structures and transmitter systems implicated in tinnitus development and provide an extensive overview on preclinical drug-based therapeutic approaches that have been explored in various animal models. As the special extract from Ginkgo biloba leaves EGb 761® has been the most widely tested drug in both non-clinical tinnitus models as well as in clinical trials, a special focus will be given to EGb 761®. The efficacy of terpene lactones, flavone glycosides and proanthocyanidines with their distinct contribution to the overall efficacy profile of the multi-constituent drug EGb 761® will be discussed.
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Ginkgo biloba , Acúfeno , Estimulación Acústica , Animales , Extractos Vegetales/uso terapéutico , Acúfeno/tratamiento farmacológicoRESUMEN
Aged rodents have been used as preclinical models of age-associated cognitive decline. Most of those models displayed substantial impairments in learning and memory. The initial, more subtle changes that precede more severe losses in cognitive abilities have not been well characterized. Here, we established a model detecting initial subtle cognitive changes by comparing the performance of moderately aged Oncins France Strain A Sprague Dawley rats with young rats in the Morris water maze (MWM) and the Open Field (OF) test. Both age groups improved their performance during the training period at a similar rate; however, the older rats performed worse in several parameters measured in the MWM. Our results suggest that already at the age of 18-20 months rats show changes in their approach to solve the spatial memory task while their ability to learn is not yet diminished. The disparate spatial information processing of the moderately aged rats provides a novel animal model for early age-related cognitive alterations that could be useful to test the effect of early intervention strategies. Moreover, our results suggest that the sensitivity of cognitive tests in the elderly could be substantially enhanced if they assess both the improvement after several trials, and the strategy used to solve a certain task.
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Cognición , Memoria Espacial , Anciano , Animales , Humanos , Aprendizaje por Laberinto , Pruebas Neuropsicológicas , Ratas , Ratas Sprague-DawleyRESUMEN
Sortilin was first identified based on its activity as part of intracellular protein sorting machinery. Recently, it was discovered that sortilin also acts as a cell surface receptor for the propeptide form of nerve growth factor (proNGF), progranulin, and neurotensin. The interaction of sortilin to these neurotrophic ligands is linked to diseases of the nervous system that lead to neurodegeneration and neuropathic pain. Blocking of the interaction of sortilin to these ligands may prevent or slow the progress of these nervous system disorders. In vitro screening assays for blocking compounds or peptides are part of the standard set of tools for drug discovery. However, assays for sortilin biology are not readily available to determine if the selected blocking agent inhibits sortilin activity on the surface of cells. We have developed a sortilin specific cell based assay to identify compounds that specifically block interaction between sortilin and proNGF prodomain. The assay system records both the presence of sortilin on the cell surface and the interaction with the pro domain of NGF. Fluorescent images of the sortilin expressing cells are analyzed for the presence of pro domain of NGF. Sortilin-positive and sortilin-negative cells within one well are concomitantly and automatically analyzed. Sortilin-pro domain interaction can be blocked dose dependently by neurotensin and synthetic compounds. The assay will facilitate the discovery of entities interfering with the binding of sortilin to the NGF pro domain. This assay can be modified to screen for inhibitors of the binding of ligands to other complex cell surface receptors.
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Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Bioensayo , Factor de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Bioensayo/métodos , Fenómenos Biofísicos , Humanos , Ligandos , Glicoproteínas de Membrana/metabolismo , Neuronas/metabolismo , Precursores de Proteínas/metabolismoRESUMEN
GPR139 is an orphan G protein-coupled receptor expressed mainly in the central nervous system. We developed a pharmacophore model based on known GPR139 surrogate agonists which led us to propose aromatic-containing dipeptides as potential ligands. Upon testing, the dipeptides demonstrated agonism in the Gq pathway. Next, in testing all 20 proteinogenic l-α-amino acids, L-tryptophan and l-phenylalanine were found to have EC50 values of 220 and 320 µM, respectively, making them the first putative endogenous agonists for GPR139.
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Aminoácidos Aromáticos/química , Aminoácidos Aromáticos/farmacología , Dipéptidos/química , Dipéptidos/farmacología , Diseño de Fármacos , Proteínas del Tejido Nervioso/agonistas , Receptores Acoplados a Proteínas G/agonistas , Diseño Asistido por Computadora , Células HEK293 , Humanos , Modelos Moleculares , Proteínas del Tejido Nervioso/metabolismo , Fenilalanina/química , Fenilalanina/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Triptófano/química , Triptófano/farmacologíaRESUMEN
Novel therapeutic concepts against cerebral ischemia focus on cell-based therapies in order to overcome some of the side effects of thrombolytic therapy. However, cell-based therapies are hampered because of restricted understanding regarding optimal cell transplantation routes and due to low survival rates of grafted cells. We therefore transplanted adult green fluorescence protein positive neural precursor cells (NPCs) either intravenously (systemic) or intrastriatally (intracerebrally) 6 hours after stroke in mice. To enhance survival of NPCs, cells were in vitro protein-transduced with TAT-heat shock protein 70 (Hsp70) before transplantation followed by a systematic analysis of brain injury and underlying mechanisms depending on cell delivery routes. Transduction of NPCs with TAT-Hsp70 resulted in increased intracerebral numbers of grafted NPCs after intracerebral but not after systemic transplantation. Whereas systemic delivery of either native or transduced NPCs yielded sustained neuroprotection and induced neurological recovery, only TAT-Hsp70-transduced NPCs prevented secondary neuronal degeneration after intracerebral delivery that was associated with enhanced functional outcome. Furthermore, intracerebral transplantation of TAT-Hsp70-transduced NPCs enhanced postischemic neurogenesis and induced sustained high levels of brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and vascular endothelial growth factor in vivo. Neuroprotection after intracerebral cell delivery correlated with the amount of surviving NPCs. On the contrary, systemic delivery of NPCs mediated acute neuroprotection via stabilization of the blood-brain-barrier, concomitant with reduced activation of matrix metalloprotease 9 and decreased formation of reactive oxygen species. Our findings imply two different mechanisms of action of intracerebrally and systemically transplanted NPCs, indicating that systemic NPC delivery might be more feasible for translational stroke concepts, lacking a need of in vitro manipulation of NPCs to induce long-term neuroprotection.
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Isquemia Encefálica/terapia , Proteínas HSP70 de Choque Térmico/genética , Células-Madre Neurales/fisiología , Células-Madre Neurales/trasplante , Accidente Cerebrovascular/terapia , Animales , Isquemia Encefálica/metabolismo , Isquemia Encefálica/cirugía , Diferenciación Celular/fisiología , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP70 de Choque Térmico/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Trasplante de Células Madre/métodos , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/cirugía , Transducción GenéticaRESUMEN
It is an often-neglected fact that extracts derived from the very same plant can differ significantly in their phytochemical composition, and thus also in their pharmacokinetic and pharmacodynamic properties which are the basis for their clinical efficacy and safety. The Ginkgo biloba L. [Ginkgoaceae] special extract EGb 761® is one of the best-studied plant extracts in the world. In the present review, using that extract as a paradigm, we describe insights how climate, the harvest region, processing of the plant material, the drying process, the extraction solvents, and the details of the subsequent process steps substantially impact the quality and uniformity of the final extract. We highlight the importance of regulating active constituent levels and consistent reduction of undesired substances in herbal extracts. This is accomplished by a controlled production process and corresponding analytical specifications. In conclusion, since extracts derived from the same plant can have very different phytochemical compositions, results from pharmacological, toxicological and clinical studies gained with one specific extract cannot be extrapolated to other extracts that were generated using different production processes. We propose that the heterogenous nature of extracts should be meticulously considered when evaluating the efficacy and safety of plant-derived remedies.
RESUMEN
BACKGROUND Cognitive symptoms persisting longer than 3 months after infection, such as memory loss, or difficulties concentrating, have been reported in up to one-third of patients after COVID-19. Evidence-based therapeutic interventions to treat post-COVID-19 symptoms (also called "Long-COVID symptoms") have not yet been established, and the treating physicians must rely on conjecture to help patients. Based on its mechanism of action and its efficacy in treating cognitive impairment, as well as its good tolerability, the Ginkgo biloba special extract EGb 761 has been suggested as a remedy to alleviate cognitive post-COVID-19 symptoms. In many studies, EGb 761 has been demonstrated to protect endothelial cells, to have potent anti-inflammatory effects, and to enhance neuroplasticity. CASE REPORT Here, we report for the first time the application of EGb 761 in the therapy of post-COVID-19-related cognitive deficits. Three women and 2 men, aged 26 to 59 years (average age 34.6 years), presented with concentration and attention deficits, cognitive deficiencies, and/or fatigue 9-35 weeks after infection. A daily dose of 2×80 mg of EGb 761 did not cause any detectable adverse effects, and it substantially improved or completely restored cognitive deficits and, when initially present, also other symptoms, such as fatigue and hyposmia, within an observation period of up to 6 months. CONCLUSIONS Our observations support the hypothesis that EGb 761 might be a low-risk treatment option for post-COVID-19 patients with cognitive symptoms. Moreover, we derive recommendations for randomized controlled clinical trials to confirm efficacy in that indication.
Asunto(s)
COVID-19 , Disfunción Cognitiva , Adulto , Antiinflamatorios/uso terapéutico , COVID-19/complicaciones , Cognición , Disfunción Cognitiva/tratamiento farmacológico , Disfunción Cognitiva/etiología , Células Endoteliales , Fatiga , Femenino , Ginkgo biloba , Humanos , Masculino , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Síndrome Post Agudo de COVID-19RESUMEN
Neural precursor cells (NPC) are an interesting tool in experimental stroke research, but their therapeutic potential is limited due to poor long-term survival. We therefore in vitro transduced subventricular zone-(SVZ)-derived NPC with the anti-apoptotic fusion protein TAT-Bcl-x(L) and analyzed NPC survival, differentiation, and post-stroke functional deficits after experimental ischemia in mice. Survival of TAT-Bcl-x(L)-transduced NPC, which were injected at day 7 post-stroke into the ischemic striatum, was significantly increased at 4 weeks after stroke. Increased survival of NPC was associated with reduced infarct injury and decreased post-stroke functional deficits. Animals grafted with TAT-Bcl-x(L)-transduced NPC showed an increased number of immature cells expressing the neuronal marker doublecortin. Since mature neuronal differentiation of NPC was not observed, reduced post-stroke injury cannot be attributed to enhanced neuronal regeneration, but rather to indirect by-stander effects of grafted NPC. In line with this, NPC-mediated neuroprotection of cortical neurons in vitro was associated with increased secretion of growth factors. Thus, in vitro transduction of cultivated NPC with TAT-Bcl-x(L) results in enhanced resistance of transplanted NPC followed by long-term neuroprotection and ameliorated functional deficits after transient focal cerebral ischemia in mice.
Asunto(s)
Isquemia Encefálica/genética , Isquemia Encefálica/terapia , Células-Madre Neurales/trasplante , Trasplante de Células Madre/métodos , Accidente Cerebrovascular/terapia , Transducción Genética/métodos , Proteína bcl-X/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Animales , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Isquemia Encefálica/cirugía , Supervivencia Celular/genética , Citoprotección/genética , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Recuperación de la Función/genética , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/patología , Tiempo , Proteína bcl-X/uso terapéutico , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/uso terapéuticoRESUMEN
CNS regeneration is limited by lesion-induced neuronal apoptosis and an environment inhibiting axonal elongation. Inhibition of ROCK has been previously shown to promote regeneration in retinal ganglion cells (RGC) whereas Cdk5 inhibition mainly promoted survival. Therefore, we have evaluated the effects of combined treatment with inhibitors of ROCK and Cdk5. We show that in vitro, the co-application of the Cdk5 inhibitor, Indolinone A, and the ROCK inhibitor, Y-27632, potentiated the survival-promoting effect of either substance alone. However, neurite outgrowth in vitro was promoted only by the presence of Y-27632, not by Indolinone A alone. In the ex vivo explant and the in vivo optic nerve crush model the combination of both inhibitors significantly increased neurite outgrowth at small distances, but this effect leveled off for longer neurites. In summary, the combined treatment with the Cdk5 inhibitor Indolinone A and the ROCK inhibitor Y-27632 results in a strong additive effect on neuronal survival, but is not able to increase the regenerative response beyond the effect of the ROCK inhibitor.
Asunto(s)
Supervivencia Celular/fisiología , Quinasa 5 Dependiente de la Ciclina/metabolismo , Regeneración Nerviosa/fisiología , Células Ganglionares de la Retina/enzimología , Células Ganglionares de la Retina/fisiología , Quinasas Asociadas a rho/metabolismo , Amidas/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Indoles/farmacología , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Compresión Nerviosa , Regeneración Nerviosa/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Neuritas/fisiología , Neuritas/ultraestructura , Fosfotransferasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piridinas/farmacología , Ratas , Ratas Wistar , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/efectos de los fármacos , Factor de Transcripción STAT3/metabolismo , Tubulina (Proteína)/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2), such as the G2019S mutation, are the most common cause of familial Parkinson's disease (PD). The G2019S mutation impairs neurite outgrowth. We hypothesized that those effects could be related to an altered expression of pluripotency genes, which may provide a readout for a screening assay based on LRRK2 function. Here, we show that the G2019S mutation mediates a sustained aberrant upregulation of the transcription factors Nanog and Oct4 that in wild-type are downregulated after differentiation. The aberrant regulation of Nanog can be concentration dependently reversed by LRRK2 tool inhibitors. Building on this knowledge, we developed an assay for the identification and assessment of compounds that inhibit the aberrant pathophysiological activity of mutant LRRK2. Furthermore, the aberrant neural pluripotency is consistent with Parkinson's pathophysiology and with the epidemiological association between the G2019S genotype and cancer risk.
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Ensayos de Selección de Medicamentos Antitumorales/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Diferenciación Celular/genética , Línea Celular Tumoral , Expresión Génica , Técnicas de Silenciamiento del Gen , Ensayos Analíticos de Alto Rendimiento , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Mutación , Proteína Homeótica Nanog/genética , Neuritas/metabolismo , Neuronas/citología , Neuronas/metabolismo , Regiones Promotoras GenéticasRESUMEN
Cerebral ischemia activates endogenous neurogenesis in the subventricular zone (SVZ) and the dentate gyrus. Consecutively, SVZ-derived neural precursors migrate towards ischemic lesions. However, functional relevance of activated neurogenesis is limited by poor survival of new-born precursors. We therefore employed the HI-virus-derived fusion protein TAT-Bcl-x(L) to study the effects of acute anti-apoptotic treatment on endogenous neurogenesis and functional outcome after transient cerebral ischemia in mice. TAT-Bcl-x(L) treatment led to significantly reduced acute ischemic cell death (128+/-23 vs. 305+/-65 TUNEL+ cells/mm(2) in controls) and infarct volumes resulting in less motor deficits and improved spatial learning. It significantly increased survival of doublecortin (Dcx)-positive neuronal precursors (389+/-96 vs. 213+/-97 Dcx+ cells in controls) but did not enhance overall post-ischemic cell proliferation or lesion-specific neuronal differentiation 28 days after ischemia. Our data demonstrate that post-stroke TAT-Bcl-x(L)-treatment results in acute neuroprotection, improved functional outcome, and enhanced survival of lesion-specific neuronal precursor cells after cerebral ischemia in mice.
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Isquemia Encefálica/tratamiento farmacológico , Cuerpo Estriado/fisiopatología , Productos del Gen tat/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Células Madre/fisiología , Proteína bcl-X/uso terapéutico , Animales , Isquemia Encefálica/fisiopatología , Muerte Celular/fisiología , Supervivencia Celular/fisiología , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Aprendizaje , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Actividad Motora , Neurogénesis/fisiología , Neuropéptidos/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , Percepción EspacialRESUMEN
BACKGROUND: The hematopoietic Granulocyte-Colony Stimulating Factor (G-CSF) plays a crucial role in controlling the number of neutrophil progenitor cells. Its function is mediated via the G-CSF receptor, which was recently found to be expressed also in the central nervous system. In addition, G-CSF provided neuroprotection in models of neuronal cell death. Here we used the retinal ganglion cell (RGC) axotomy model to compare effects of local and systemic application of neuroprotective molecules. RESULTS: We found that the G-CSF receptor is robustly expressed by RGCs in vivo and in vitro. We thus evaluated G-CSF as a neuroprotectant for RGCs and found a dose-dependent neuroprotective effect of G-CSF on axotomized RGCs when given subcutaneously. As stem stell mobilization had previously been discussed as a possible contributor to the neuroprotective effects of G-CSF, we compared the local treatment of RGCs by injection of G-CSF into the vitreous body with systemic delivery by subcutaneous application. Both routes of application reduced retinal ganglion cell death to a comparable extent. Moreover, G-CSF enhanced the survival of immunopurified RGCs in vitro. CONCLUSION: We thus show that G-CSF neuroprotection is at least partially independent of potential systemic effects and provide further evidence that the clinically applicable G-CSF could become a treatment option for both neurodegenerative diseases and glaucoma.
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Factor Estimulante de Colonias de Granulocitos/fisiología , Degeneración Nerviosa/tratamiento farmacológico , Fármacos Neuroprotectores/administración & dosificación , Traumatismos del Nervio Óptico/tratamiento farmacológico , Células Ganglionares de la Retina/efectos de los fármacos , Análisis de Varianza , Animales , Apoptosis/efectos de los fármacos , Axotomía , Relación Dosis-Respuesta a Droga , Femenino , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Inyecciones Subcutáneas , Ratas , Ratas Sprague-Dawley , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Células Ganglionares de la Retina/metabolismo , Estadísticas no Paramétricas , Cuerpo Vítreo/efectos de los fármacosRESUMEN
Parkinson's disease is characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra. The heat-shock protein 70 (Hsp70) reduces protein misfolding and aggregation. It has been shown to protect cells against oxidative stress and apoptotic stimuli in various neurodegenerative disease models. To deliver Hsp70 across cellular membranes and into the brain, we linked it to a cell-penetrating peptide derived from the HIV trans-activator of transcription (Tat) protein. In vitro, Tat-Hsp70 transduced neuroblastoma cells and protected primary mesencephalic DA neurons and their neurites against MPP+-mediated degeneration. In vivo, the systemic application of cell-permeable Hsp70 protected DA neurons of the substantia nigra pars compacta against subacute toxicity of MPTP. Furthermore, Tat-Hsp70 diminished the MPTP induced decrease in DA striatal fiber density. Thus, we demonstrate that systemically applied Tat-Hsp70 effectively prevents neuronal cell death in in vitro and in vivo models of Parkinson's disease. The use of Tat-fusion proteins might therefore be a valuable tool to deliver molecular chaperones like Hsp70 into the brain and may be the starting point for new protective strategies in neurodegenerative diseases.
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Productos del Gen tat/genética , Terapia Genética/métodos , Proteínas HSP70 de Choque Térmico/metabolismo , Estrés Oxidativo/genética , Proteínas Recombinantes de Fusión/metabolismo , Sustancia Negra/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Cuerpo Estriado/fisiopatología , Citoprotección/genética , Dopamina/metabolismo , Productos del Gen tat/química , Proteínas HSP70 de Choque Térmico/genética , Humanos , Masculino , Ratones , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/terapia , Fragmentos de Péptidos/genética , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/uso terapéutico , Sustancia Negra/citología , Sustancia Negra/fisiopatología , Transducción Genética/métodos , Resultado del Tratamiento , Degeneración Walleriana/genética , Degeneración Walleriana/metabolismo , Degeneración Walleriana/terapiaRESUMEN
The anti-apoptotic Bcl-xL is a promising agent to prevent neurodegeneration in Parkinson's disease, which is characterized by a demise of dopaminergic neurons. We linked Bcl-xL to a peptide that allows its delivery across biological membranes and the blood-brain barrier. We tested the fusion protein in two models of Parkinson's Disease. Cell-permeable Bcl-xL protected neuroblastoma cells from the selective neurotoxin 1-methyl-4-phenylpyridinium. Furthermore, its systemic application in aged mice protected dopaminergic neurons following administration of MPTP as revealed by counting of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra pars compacta. Hence, we present that a cell-permeable form of an anti-apoptotic protein can be delivered to CNS neurons through its systemic application, and we provide the proof that the delivery of this protein to the CNS neurons effectively prevents neuronal cell death in models of chronic neurodegenerative diseases.
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1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Dopamina/metabolismo , Neuronas/efectos de los fármacos , Neurotoxinas/farmacología , Sustancia Negra/patología , Proteína bcl-X/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Neuroblastoma , Neurotoxinas/metabolismo , Enfermedad de Parkinson/patología , Proteínas Recombinantes/metabolismo , Sustancia Negra/efectos de los fármacosRESUMEN
Cell-penetrating peptides (CPPs), such as the one derived from the human immunodeficiency virus Tat protein, facilitate the delivery of cargoes across cellular membranes. However, questions about the therapeutic potential of CPP-mediated delivery remain. For instance, the impact of the purification procedure on the functionality of Tat-fusion proteins has not been systematically examined. Here, we isolated fusion proteins of the chaperone heat shock protein 70 (Hsp70) and the Tat CPP under denaturing or native conditions. To investigate the therapeutic potential of different recombinant protein preparations, we examined Tat-Hsp70 transduction efficiency and quantified Tat-Hsp70-mediated folding of a chaperone-dependent yellow fluorescent protein in vitro. Transduction efficiency and chaperone activity of Tat-Hsp70-treated cells was significantly higher compared to cells treated with Hsp70. The application of native isolated Tat-Hsp70 had the strongest effect. This chaperone activity correlates with increased viability of cells treated with the recombinant protein after cell death induction with 6-hydroxydopamine. This suggests that the method of recombinant Tat-fusion protein purification influences its functionality. For Tat-Hsp70, the method of choice seems to be isolation under native conditions, for which we present a purification protocol. Our results may contribute to improve Tat-fusion protein application in basic research and may facilitate its use as therapeutic tool, for instance in Parkinson's disease.
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Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/farmacología , Animales , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Productos del Gen tat/genética , Productos del Gen tat/metabolismo , Productos del Gen tat/farmacología , Proteínas HSP70 de Choque Térmico/genética , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Neuroblastoma , Oxidopamina/toxicidad , Transporte de Proteínas , Ratas , Proteínas Recombinantes/metabolismo , Simpaticolíticos/toxicidad , TransfecciónRESUMEN
BACKGROUND: Neuroinflammation is a key factor of Alzheimer's disease (AD) and other neurodegenerative conditions. Microglia are the resident mononuclear immune cells of the central nervous system (CNS). They play an essential role in the maintenance of homeostasis and responses to neuroinflammation. Ginkgo biloba extract EGb 761 is one of the most commonly used natural medicines owing to its established efficacy and remarkable biological activities especially in respect to CNS diseases. However, only few studies have addressed the effects and mechanisms of Ginkgo biloba extract in microglia activation. METHODS: We measured the production of pro-inflammatory mediators and cytokines by ELISA and analyzed gene expressions by qRT-PCR and Western Blot in LPS treated cultured primary rat microglia. RESULTS: The Ginkgo biloba extract EGb 761 significantly inhibited the release of prostaglandin E2 (PGE2) and differentially regulated the levels of pro-inflammatory cytokines. The inhibition of LPS-induced PGE2 release in primary microglia was partially dependent on reduced protein synthesis of mPGES-1 and the reduction in the activation of cytosolic phospholipase A2 (cPLA2) without altering COX-2 enzymatic activity, inhibitor of kappa B alpha (IkappaBalpha) degradation, and the activation of multiple mitogen activated protein kinases (MAPKs). Altogether, we showed that EGb 761 reduces neuro-inflammatory activation in primary microglial cells by targeting PGE2 release and cytokines. CONCLUSION: Ginkgo biloba extract EGb 761 displayed anti-neuroinflammatory activity in LPS-activated primary microglia cells. EGb 761 was able to reduce neuroinflammatory activation by targeting the COX/PGE2 pathway. This effect might contribute to the established clinical cognitive efficacy in Alzheimer's disease, vascular and mixed dementia.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Microglía/efectos de los fármacos , Extractos Vegetales/farmacología , Animales , Células Cultivadas , Ciclooxigenasa 2/metabolismo , Citocinas/metabolismo , Dinoprost/análogos & derivados , Dinoprost/metabolismo , Dinoprostona/metabolismo , Ginkgo biloba , Fosfolipasas A2 Grupo IV/metabolismo , Proteínas I-kappa B/antagonistas & inhibidores , Proteínas I-kappa B/metabolismo , Lipopolisacáridos/farmacología , Microglía/metabolismo , Microglía/patología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , RatasRESUMEN
Many aspects of stress-induced physiological and psychological effects have been characterized in people and animals. However, stress effects on the auditory system are less explored and their mechanisms are not well-understood, in spite of its relevance for a variety of diseases, including tinnitus. To expedite further research of stress-induced changes in the auditory system, here we compare the reactions to stress among Wistar and Lewis rats. The animals were stressed for 24 h, and subsequently we tested the functionality of the outer hair cells (OHCs) using distortion product otoacoustic emissions (DPOAEs) and auditory neurons using evoked auditory brainstem responses (ABR). Lastly, using Western blot, we analyzed the levels of plasticity-related proteins in the inferior colliculus, confirming that the inferior colliculus is involved in the adaptive changes that occur in the auditory system upon stress exposure. Surprisingly, the two strains reacted to stress quite differently: Lewis rats displayed a lowering of their auditory threshold, whereas it was increased in Wistar rats. These functional differences were seen in OHCs of the apical region (low frequencies) and in the auditory neurons (across several frequencies) from day 1 until 2 weeks after the experimental stress ended. Wistar and Lewis rats may thus provide models for auditory threshold increase and decrease, respectively, which can both be observed in different patients in response to stress.
RESUMEN
Neuronal death can take on many different forms, from well-defined apoptosis to caspase-independent processes. While members of the Bcl-2 family of intracellular proteins are known to be involved in classic apoptotic cascades, their role in necrosis has been less well defined. Here, we applied a cell-permeable form of the anti-apoptotic Bcl-2 family member Bcl-x(L) on glutamate-treated rat primary cerebellar granule neurons to test its effect on neuronal survival. Bcl-x(L) inhibited the late phase of cell death, when caspases are activated, but it did not inhibit the early, caspase-independent phase of cell death. These different phases of cell death following glutamate treatment have not been taken into account in many earlier reports either supporting or refuting an involvement of Bcl-2 family members in excitotoxic cell death. Our results suggest that under our experimental conditions, Bcl-x(L) inhibits caspase-dependent apoptosis, but not caspase-independent neuronal death.