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1.
Int J Mol Sci ; 24(11)2023 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-37298488

RESUMEN

Regulated systems for transgene expression are useful tools in basic research and a promising platform in biomedicine due to their regulated transgene expression by an inducer. The emergence of optogenetics expression systems enabled the construction of light-switchable systems, enhancing the spatial and temporal resolution of a transgene. The LightOn system is an optogenetic tool that regulates the expression of a gene of interest using blue light as an inducer. This system is based on a photosensitive protein (GAVPO), which dimerizes and binds to the UASG sequence in response to blue light, triggering the expression of a downstream transgene. Previously, we adapted the LightOn system to a dual lentiviral vector system for neurons. Here, we continue the optimization and assemble all components of the LightOn system into a single lentiviral plasmid, the OPTO-BLUE system. For functional validation, we used enhanced green fluorescent protein (EGFP) as an expression reporter (OPTO-BLUE-EGFP) and evaluated the efficiency of EGFP expression by transfection and transduction in HEK293-T cells exposed to continuous blue-light illumination. Altogether, these results prove that the optimized OPTO-BLUE system allows the light-controlled expression of a reporter protein according to a specific time and light intensity. Likewise, this system should provide an important molecular tool to modulate gene expression of any protein by blue light.


Asunto(s)
Vectores Genéticos , Optogenética , Humanos , Optogenética/métodos , Células HEK293 , Transfección , Transgenes , Expresión Génica , Vectores Genéticos/genética , Lentivirus/genética
2.
Cells ; 11(9)2022 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-35563850

RESUMEN

Parkinson's disease (PD) is a neurodegenerative disease characterized by motor symptoms and dopaminergic cell loss. A pre-symptomatic phase characterized by non-motor symptoms precedes the onset of motor alterations. Two recent PET studies in human carriers of mutations associated with familial PD demonstrate an early serotonergic commitment-alteration in SERT binding-before any dopaminergic or motor dysfunction, that is, at putative PD pre-symptomatic stages. These findings support the hypothesis that early alterations in the serotonergic system could contribute to the progression of PD, an idea difficult to be tested in humans. Here, we study some components of the serotonergic system during the pre-symptomatic phase in a well-characterized Drosophila PD model, Pink1B9 mutant flies. We detected lower brain serotonin content in Pink1B9 flies, accompanied by reduced activity of SERT before the onset of motor dysfunctions. We also explored the consequences of a brief early manipulation of the serotonergic system in the development of motor symptoms later in aged animals. Feeding young Pink1B9 flies with fluoxetine, a SERT blocker, prevents the loss of dopaminergic neurons and ameliorates motor impairment observed in aged mutant flies. Surprisingly, the same pharmacological manipulation in young control flies results in aged animals exhibiting a PD-like phenotype. Our findings support that an early dysfunction in the serotonergic system precedes and contributes to the onset of the Parkinsonian phenotype in Drosophila.


Asunto(s)
Proteínas de Drosophila , Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Animales , Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Enfermedad de Parkinson/genética , Fenotipo , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Transmisión Sináptica
3.
Sci Rep ; 11(1): 9579, 2021 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-33953275

RESUMEN

It has been observed that there is a lower Parkinson's disease (PD) incidence in tobacco users. Nicotine is a cholinergic agonist and is the principal psychoactive compound in tobacco linked to cigarette addiction. Different studies have shown that nicotine has beneficial effects on sporadic and genetic models of PD. In this work we evaluate nicotine's protective effect in a Drosophila melanogaster model for PD where Synphilin-1 (Sph-1) is expressed in dopaminergic neurons. Nicotine has a moderate effect on dopaminergic neuron survival that becomes more evident as flies age. Nicotine is beneficial on fly survival and motility increasing tyrosine hydroxylase and dopamine levels, suggesting that cholinergic agonists may promote survival and metabolic function of the dopaminergic neurons that express Sph-1. The Sph-1 expressing fly is a good model for the study of early-onset phenotypes such as olfaction loss one of the main non-motor symptom related to PD. Our data suggest that nicotine is an interesting therapeutic molecule whose properties should be explored in future research on the phenotypic modulators of the disease and for the development of new treatments.


Asunto(s)
Proteínas Portadoras/metabolismo , Dopamina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Nicotina/uso terapéutico , Enfermedad de Parkinson/tratamiento farmacológico , Tirosina 3-Monooxigenasa/metabolismo , Animales , Proteínas Portadoras/genética , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Drosophila melanogaster , Ratones , Proteínas del Tejido Nervioso/genética , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Agonistas Nicotínicos/uso terapéutico , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo
4.
Electron. j. biotechnol ; 51: 50-57, May. 2021. ilus, graf
Artículo en Inglés | LILACS | ID: biblio-1343384

RESUMEN

BACKGROUND: Molecular brain therapies require the development of molecular switches to control gene expression in a limited and regulated manner in time and space. Light-switchable gene systems allow precise control of gene expression with an enhanced spatio-temporal resolution compared to chemical inducers. In this work, we adapted the existing light-switchable Light-On system into a lentiviral platform, which consists of two modules: (i) one for the expression of the blue light-switchable transactivator GAVPO and (ii) a second module containing an inducible-UAS promoter (UAS) modulated by a light-activated GAVPO. RESULTS: In the HEK293-T cell line transfected with this lentiviral plasmids system, the expression of the reporter mCherry increased between 4 to 5 fold after light induction. A time expression analysis after light induction during 24 h revealed that mRNA levels continuously increased up to 9 h, while protein levels increased throughout the experiment. Finally, transduction of cultured rat hippocampal neurons with this dual Light-On lentiviral system showed that CDNF, a potential therapeutic trophic factor, was induced only in cells exposed to blue light. CONCLUSIONS: In conclusion, the optimized lentiviral platform of the Light-On system provides an efficient way to control gene expression in neurons, suggesting that this platform could potentially be used in biomedical and neuroscience research, and eventually in brain therapies for neurodegenerative diseases.


Asunto(s)
Regulación de la Expresión Génica , Optogenética/métodos , Luz , Neuronas/metabolismo , Immunoblotting , Expresión Génica , Técnica del Anticuerpo Fluorescente , Lentivirus
5.
Neurochem Int ; 138: 104753, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32416114

RESUMEN

Mutations in the dystrobrevin binding protein 1 (DTNBP1) gene that encodes for the dysbindin-1 protein, are associated with a higher risk for schizophrenia. Interestingly, individuals carrying high-risk alleles in this gene have been associated with an increased incidence of negative symptoms for the disease, which include anhedonia, avolition and social withdrawal. Here we evaluated behavioral and neurochemical changes in a hypomorphic Drosophila mutant for the orthologue of human Dysbindin-1, dysb1. Mutant dysb1 flies exhibit altered social space parameters, suggesting asocial behavior, accompanied by reduced olfactory performance. Moreover, dysb1 mutant flies show poor performance in basal and startle-induced locomotor activity. We also report a reduction in serotonin brain levels and changes in the expression of the Drosophila serotonin transporter (dSERT) in dysb1 flies. Our data show that the serotonin-releasing amphetamine derivative 4-methylthioamphetamine (4-MTA) modulates social spacing and locomotion in control flies, suggesting that serotonergic circuits modulate these behaviors. 4-MTA was unable to modify the behavioral deficiencies in mutant flies, which is consistent with the idea that the efficiency of pharmacological agents acting at dSERT depends on functional serotonergic circuits. Thus, our data show that the dysb1 mutant exhibits behavioral deficits that mirror some aspects of the endophenotypes associated with the negative symptoms of schizophrenia. We argue that at least part of the behavioral aspects associated with these symptoms could be explained by a serotonergic deficit. The dysb1 mutant presents an opportunity to study the molecular underpinnings of schizophrenia negative symptoms and reveals new potential targets for treatment of the disease.


Asunto(s)
Proteínas de Drosophila/genética , Disbindina/genética , Mutación/genética , Esquizofrenia/genética , Serotonina/genética , Interacción Social , Animales , Animales Modificados Genéticamente , Drosophila , Proteínas de Drosophila/metabolismo , Disbindina/metabolismo , Humanos , Masculino , Esquizofrenia/metabolismo , Serotonina/metabolismo , Olfato/fisiología
6.
Biochim Biophys Acta Mol Basis Dis ; 1863(11): 2882-2890, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28716706

RESUMEN

Parkinson's disease (PD) is a degenerative disorder characterized by several motor symptoms including shaking, rigidity, slow movement and difficult walking, which has been associated to the death of nigro-striatal dopaminergic neurons. >90% of PD patients also present olfactory dysfunction. Although the molecular mechanisms responsible for this disease are not clear, hereditary PD is linked to mutations in specific genes, including the PTEN-induced putative kinase 1 (PINK1). In this work we provide for the first time a thorough temporal description of the behavioral effects induced by a mutation in the PINK1 gene in adult Drosophila, a previously described animal model for PD. Our data suggests that the motor deficits associated to PD are fully revealed only by the third week of age. However, olfactory dysfunction is detected as early as the first week of age. We also provide immunofluorescence and neurochemical data that let us propose for the first time the idea that compensatory changes occur in this Drosophila model for PD. These compensatory changes are associated to specific components of the dopaminergic system: the biosynthetic enzymes, Tyrosine hydroxylase and Dopa decarboxylase, and the Dopamine transporter, a plasma membrane protein involved in maintaining dopamine extracellular levels at physiologically relevant levels. Thus, our behavioral, immunofluorescence and neurochemical data help define for the first time presymptomatic and symptomatic phases in this PD animal model, and that compensatory changes occur in the dopaminergic neurons in the presymptomatic stage.


Asunto(s)
Conducta Animal , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Enfermedad de Parkinson/metabolismo , Animales , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/patología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/fisiopatología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo
7.
ACS Chem Neurosci ; 8(10): 2168-2179, 2017 10 18.
Artículo en Inglés | MEDLINE | ID: mdl-28665105

RESUMEN

A better comprehension on how different molecular components of the serotonergic system contribute to the adequate regulation of behaviors in animals is essential in the interpretation on how they are involved in neuropsychiatric and pathological disorders. It is possible to study these components in "simpler" animal models including the fly Drosophila melanogaster, given that most of the components of the serotonergic system are conserved between vertebrates and invertebrates. Here we decided to advance our understanding on how the serotonin plasma membrane transporter (SERT) contributes to serotonergic neurotransmission and behaviors in Drosophila. In doing this, we characterized for the first time a mutant for Drosophila SERT (dSERT) and additionally used a highly selective serotonin-releasing drug, 4-methylthioamphetamine (4-MTA), whose mechanism of action involves the SERT protein. Our results show that dSERT mutant animals exhibit an increased survival rate in stress conditions, increased basal motor behavior, and decreased levels in an anxiety-related parameter, centrophobism. We also show that 4-MTA increases the negative chemotaxis toward a strong aversive odorant, benzaldehyde. Our neurochemical data suggest that this effect is mediated by dSERT and depends on the 4-MTA-increased release of serotonin in the fly brain. Our in silico data support the idea that these effects are explained by specific interactions between 4-MTA and dSERT. In sum, our neurochemical, in silico, and behavioral analyses demonstrate the critical importance of the serotonergic system and particularly dSERT functioning in modulating several behaviors in Drosophila.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Serotonina/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Proteínas de Drosophila/genética , Mutación/genética , Serotoninérgicos/metabolismo , Serotoninérgicos/farmacología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética
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