RESUMEN
Background: In addition to motor disability, another characteristic feature of Parkinson disease is the early appearance of psychiatric symptoms, including apathy, depression, anxiety and cognitive deficits; treatments for these symptoms are limited by the development of adverse effects such as impulse-control disorders. In this context, we investigated the orphan G protein-coupled receptor 88 (GPR88) as a novel therapeutic target. Methods: We used lentiviral-mediated expression of specifically designed microRNA to knock down Gpr88 in a translational male rat model of early Parkinson disease obtained by dopamine loss in the dorsolateral striatum as a result of 6-hydroxydopamine lesions. We evaluated the impact of Gpr88 knockdown on the Parkinson disease model using behavioural, immunohistochemical and in situ hybridization studies. Results: Knockdown of Gpr88 in associative territories of the dorsal striatum efficiently reduced alterations in mood, motivation and cognition through modulation of the regulator of the G-protein signalling 4 and of the truncated splice variant of the FosB transcription factor. Knockdown of Gpr88 also reduced allostatic changes in striatal activity markers that may be related to patterns observed in patients and that provide support for an "overload" hypothesis for the etiology of the psychiatric symptoms of Parkinson disease. Limitations: Behavioural tests assessing specific cognitive and motivational parameters are needed to further characterize the effects of the lesion and of Gpr88 knockdown in early-stage and advanced Parkinson disease models, presenting more extensive dopamine loss. Additional studies focusing on the direct and indirect striatal output pathways are also required, because little is known about the signalling pathways regulated by GPR88 in different striatal cell types. Conclusion: GPR88 may constitute a highly relevant target for the treatment of the psychiatric symptoms of Parkinson disease.
Asunto(s)
Conducta Animal/fisiología , Síntomas Conductuales , Neostriado , Enfermedad de Parkinson , Receptores Acoplados a Proteínas G/metabolismo , Animales , Síntomas Conductuales/etiología , Síntomas Conductuales/metabolismo , Síntomas Conductuales/fisiopatología , Modelos Animales de Enfermedad , Humanos , Masculino , Neostriado/metabolismo , Neostriado/fisiopatología , Enfermedad de Parkinson/complicaciones , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/fisiopatología , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Receptores Acoplados a Proteínas G/genética , Investigación Biomédica TraslacionalRESUMEN
The effects of L-3-4-dyhydroxyphenylalanine (L-DOPA) treatment for replacing the dopamine (DA) loss in Parkinson's disease (PD) progressively wear off and are hindered by the development of dyskinesia, prompting the search for new treatments. The orphan G protein-coupled receptor 88 (Gpr88) represents a potential new target, as it is highly and almost exclusively expressed in the projecting gamma-Aminobutyric Acid-ergic (GABAergic) medium spiny neurons of the striatum, is implicated in motor activity, and is downregulated by 6-hydroxydopamine (6-OHDA) lesions, an effect that is reversed by L-DOPA. Thus, to evaluate Gpr88 as a potential target for the management of PD and L-DOPA-induced dyskinesia (LID), we inactivated Gpr88 by lentiviral-mediated knock-down with a specifically designed microRNA (miR) (KD-Gpr88) in a 6-OHDA rat model of hemiparkinsonism. Then, we investigated the effects of the KD-Gpr88 in the DA-deprived dorsal striatum on circling behavior and LID as well as on specific markers of striatal neuron activity. The KD-Gpr88 reduced the acute amphetamine-induced and increased L-DOPA-induced turning behavior. Moreover, it normalized the upregulated expression of striatal Gad67 and proenkephalin provoked by the 6-OHDA lesion. Finally, despite promoting ΔFosB accumulation, the KD-Gpr88 was associated neither with the upregulation of prodynorphin, which is causally linked to the severity of LID, nor with the aggravation of LID following chronic L-DOPA treatment in 6-OHDA-lesioned rats. These results thus justify further evaluation of Gpr88 as a potentially novel target for the management of PD as an alternative to L-DOPA therapy.
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Aminoácidos Esenciales/deficiencia , Alimentos Formulados , Terapia Genética/métodos , Elementos de Respuesta , Transgenes , Adulto , Aminoácidos Esenciales/farmacología , Animales , Proteínas de Ciclo Celular/genética , Pollos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Ratas , Proteínas Represoras/genética , Elementos de Respuesta/efectos de los fármacos , Porcinos , Timidina Quinasa/genética , Activación Transcripcional/efectos de los fármacos , Transgenes/efectos de los fármacos , Transgenes/genéticaRESUMEN
Widespread application of gene therapy will depend on the development of simple methods to regulate the expression of therapeutic genes. Here we harness an endogenous signaling pathway to regulate therapeutic gene expression through diet. The GCN2-eIF2α signaling pathway is specifically activated by deficiencies in any essential amino acid (EAA); EAA deficiency leads to rapid expression of genes regulated by ATF4-binding cis elements. We found that therapeutic genes under the control of optimized amino acid response elements (AAREs) had low basal expression and high induced expression. We applied our system to regulate the expression of TNFSF10 (TRAIL) in the context of glioma therapy and found that intermittent activation of this gene by EEA-deficient meals retained its therapeutic efficacy while abrogating its toxic effects on normal tissue. The GCN2-eIF2α pathway is expressed in many tissues, including the brain, and is highly specific to EAA deficiency. Our system may be particularly well suited for intermittent regulation of therapeutic transgenes over short or long time periods.
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Aminoácidos Esenciales/administración & dosificación , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Terapia Genética/métodos , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/uso terapéutico , Administración Oral , Aminoácidos Esenciales/farmacocinética , Animales , Suplementos Dietéticos , Relación Dosis-Respuesta a Droga , Ingestión de Alimentos/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Masculino , Ratones , Transgenes/genética , Resultado del TratamientoRESUMEN
The present study was intended to combine three therapeutic approaches in a well-defined rat model of spinal cord injury, a lateral hemisection at thoracic level. A guidance channel was implanted at the lesion site. This channel was seeded with native Schwann cells or Schwann cells that had been previously transduced with a lentiviral vector carrying the GDNF gene. Thereafter, these experiences were reproduced in animals injected with lentiviral vectors carrying a shRNA for GFAP (Lv-shGFAP), which has recently been shown to block glial scar formation. Functional evaluations showed that Lv-shGFAP induced a significant improvement in recovery in animals grafted with Schwann cells. Histological studies demonstrated the outgrowth of axons in the guidance channel containing Schwann cells transduced or not with GDNF. This axonal growth was enhanced in rats receiving Lv-shGFAP vector. Also, a significant increase of serotonergic innervation of the injured hemicord, distal to the lesion, was found only in animals treated with Lv-shGFAP vectors. Importantly, this study confirms that glial scar formation is a major impediment for axonal sprouting after spinal cord injury, and emphasizes the importance of serotonergic innervation for locomotor function. Moreover we show a significant additive effect of a combinatorial approach to axonal regeneration in the injured spinal cord.
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Neuroglía/patología , Células de Schwann/trasplante , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/terapia , Animales , Supervivencia Celular , Cicatriz/patología , Femenino , Vectores Genéticos , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Lentivirus/genética , Locomoción , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Recuperación de la Función , Neuronas Serotoninérgicas/fisiología , Médula Espinal/patologíaRESUMEN
In spinal cord injury (SCI), absence of functional recovery and lack of spontaneous axonal regeneration are attributed, among other factors, to the formation of a glial scar that forms both physical and chemical barriers. The glial scar is composed mainly of reactive astrocytes that overexpress two intermediate filament proteins, glial fibrillary acidic protein (GFAP) and vimentin (VIM). To promote regeneration and sprouting of spared axons after spinal cord trauma and with the objective of translation to clinics, we designed an original in vivo gene transfer strategy to reduce glial scar formation after SCI, based on the RNA interference (RNAi)-mediated inhibition of GFAP and VIM. We first show that direct injection of lentiviral vectors expressing short hairpin RNA (shRNA) against GFAP and VIM in a mouse model of SCI allows efficient and specific targeting of astrocytes. We then demonstrate that the lentiviral-mediated and stable expression of shGFAP and shVIM leads to a strong reduction of astrogliosis, improves functional motor recovery, and promotes axonal regrowth and sprouting of spared axons. This study thus examplifies how the nonneuronal environment might be a major target within the lesioned central nervous system to promote axonal regeneration (and sprouting) and validates the use of lentiviral-mediated RNAi in SCI.
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Regulación de la Expresión Génica/fisiología , Proteína Ácida Fibrilar de la Glía/metabolismo , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/terapia , Vimentina/metabolismo , Análisis de Varianza , Animales , Astrocitos/metabolismo , Axones/fisiología , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos/fisiología , Proteína Ácida Fibrilar de la Glía/genética , Lentivirus/genética , Locomoción/fisiología , Ratones , Ratones Endogámicos C57BL , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Serotonina/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Vimentina/genéticaRESUMEN
Cellular effects of ethanol in YD-15 tongue carcinoma cells were assessed by MTT assay, caspase activity assay, Western blotting and flow cytometry. Ethanol inhibited the growth and proliferation of YD-15 cells in a dose- and time-dependent manner in an MTT assay. The effects of ethanol on cell cycle control at low percent range of ethanol concentration (0 to 1.5%), the condition not inducing YD-15 cell death, was investigated after exposing cells to alcohol for a certain period of time. Western blotting on the expression of cell cycle inhibitors showed that p21 and p27 was up-regulated as ethanol concentration increases from 0 to 1.5% whilst the cell cycle regulators, cdk1, cdk2, and cdk4 as well as Cyclin A, Cyclin B1 and Cyclin E1, were gradually down-regulated. Flow cytometric analysis of cell cycle distribution revealed that YD-15 cells exposed to 1.5% ethanol for 24 h was mainly arrested at G2/M phase. However, ethanol induced apoptosis in YD-15 cells exposed to 2.5% or higher percent of ethanol. The cleaved PARP, a marker of caspase-3 mediated apoptosis, and the activation of caspase-3 and -7 were detected by caspase activity assay or Western blotting. Our results suggest that ethanol elicits inhibitory effect on the growth and proliferation of YD-15 tongue carcinoma cells by mediating cell cycle arrest at G2/M at low concentration range and ultimately induces apoptosis under the condition of high concentration.
RESUMEN
Anthocyanins are naturally occuring phytochemicals and the main components of the coloring of plants, flowers and fruits. They are known to elicit antioxidative, anti-inflammatory and cancer preventive activity. In this study, we investigated anthocyanins in black / yellow soybean seedcoats using different methods of detection - thin layer chromatography (TLC), capillary zone electrophoresis (CZE) and HPLC analysis. The anthocyanins in soybean seedcoats were extracted by five independent methods of extraction and the aglycons (anthocyanidins) of the corresponding anthocyanins were prepared by acid mediated hydrolysis. The anthocyanin / anthocyanidin in black soybean seedcoat showed characteristic TLC mobility, CZE electrophoretic retention and HPLC migration time while little of anthocyanins were detected from yellow soybean seedcoat. The extracted anthocyanins showed pH dependent retention time in CZE and spectral change in UV-Vis spectrum. HPLC analysis of the hydrolyzed extract of black soybean seedcoat identified the presence of four anthocyanidins. The major anthocyanin in black soybean seedcoat was cyanin (cyanidin-3-O-glucoside), with the relative order of anthocyanidin in cyanidin > delphinidin > petunidin > pelargonidin.