Optimization of the Light-On system in a lentiviral platform to a light-controlled expression of genes in neurons

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.

Advances in the application of optogenetic in the treatment of retinal degenerative diseases / 中华眼底病杂志

Retinal degeneration is a blind eye disease caused by changes in the function of retinal pigment epithelial cells and photoreceptor cells. Stem cell transplantation, gene therapy, retinal prosthesis implantation and other new biological technologies have made great progress in the restoration of visual function, but they still face many difficulties. Optogenetic is a new interdisciplinary technology that combines optics, physiology and genetics. It can express photosensitive proteins on retinal neurons in retinal degeneration. The light stimulation causing depolarization or hyperpolarization reaction of cells that expressed photosensitive proteins to gain light sensitivity. Compared with the immune rejection of stem cell therapy, the greater individualization of gene therapy and the greater traumatic nature of retinal prosthesis implantation, optogenetic technology has significant advantages, and it is also urgent to solve the problems of low spatial and temporal resolution and light sensitivity. With the gradual development of optogenetics technology, it is bound to form a deeper level of cross and fusion with other fields, so as to contribute to the recovery of visual function of patients with retinal degeneration.

Focus on the treatment of inherited retinal diseases / 中华实验眼科杂志

Inherited retinal diseases (IRDs) are rare and incurable eye diseases.Gene therapy has become a new method for the treatment of IRDs.At present, at least 26 clinical trials of gene therapy involving at least 16 different IRDs genes are in progress or imminent.Such as autosomal recessive retinitis pigmentosa (RP, MERTK mutation), choroideremia (CHM mutation), Stargardt’s disease (ABCA4 mutation), Usher syndrome 1B subtype (Myo7a mutation), X-linked retinoschisis (RS1 mutation), mitochondrial-related Leber hereditary optic neuropathy (ND4 mutation), panchromatic blindness (CNGA3 mutation and CNNGB3 mutation), sex-linked RP (RPGR mutation). Adeno-associated viral (AAV) vector-mediated gene expression is a conventional method for gene transduction, which has showed efficacies in autosomal recessive IRDs caused by small gene mutations, especially for those IRDs, which have the original lesions in retinal pigment epithelial (RPE) cells and photoreceptor cells.Novel progress has been made in animal experiments by gene editing technologies for autosomal dominant IRDs or autosomal recessive IRDs with large gene mutations, which is going to clinical trial.Oligonucleotide therapy based on RNA has also shown good efficacy for the IRDs caused by large genes, which beyond the AAV capacity.These research achievements are attacking more and more attentions in these fields.Although there are still some efficacy and safety issues on the way to treat IRDs, it is reasonable to expect that more and more patients with IRDs will be treated in the near future.

Focus on the treatment of inherited retinal diseases / 中华实验眼科杂志

Inherited retinal diseases ( IRDs) are rare and incurable eye diseases. Gene therapy has become a new method for the treatment of IRDs. At present, at least 26 clinical trials of gene therapy involving at least 16 different IRDs genes are in progress or imminent. Such as autosomal recessive retinitis pigmentosa ( RP, MERTK mutation),choroideremia (CHM mutation),Stargardt' s disease (ABCA4 mutation),Usher syndrome 1B subtype (Myo7a mutation),X-linked retinoschisis (RS1 mutation),mitochondrial-related Leber hereditary optic neuropathy ( ND4 mutation ) , panchromatic blindness ( CNGA3 mutation and CNNGB3 mutation ) , sex-linked RP ( RPGR mutation) . Adeno-associated viral ( AAV ) vector-mediated gene expression is a conventional method for gene transduction,which has showed efficacies in autosomal recessive IRDs caused by small gene mutations,especially for those IRDs,which have the original lesions in retinal pigment epithelial ( RPE) cells and photoreceptor cells. Novel progress has been made in animal experiments by gene editing technologies for autosomal dominant IRDs or autosomal recessive IRDs with large gene mutations,which is going to clinical trial. Oligonucleotide therapy based on RNA has also shown good efficacy for the IRDs caused by large genes, which beyond the AAV capacity. These research achievements are attacking more and more attentions in these fields. Although there are still some efficacy and safety issues on the way to treat IRDs,it is reasonable to expect that more and more patients with IRDs will be treated in the near future.

Pulse-train Stimulation of Primary Somatosensory Cortex Blocks Pain Perception in Tail Clip Test

Human studies of brain stimulation have demonstrated modulatory effects on the perception of pain. However, whether the primary somatosensory cortical activity is associated with antinociceptive responses remains unknown. Therefore, we examined the antinociceptive effects of neuronal activity evoked by optogenetic stimulation of primary somatosensory cortex. Optogenetic transgenic mice were subjected to continuous or pulse-train optogenetic stimulation of the primary somatosensory cortex at frequencies of 15, 30, and 40 Hz, during a tail clip test. Reaction time was measured using a digital high-speed video camera. Pulse-train optogenetic stimulation of primary somatosensory cortex showed a delayed pain response with respect to a tail clip, whereas no significant change in reaction time was observed with continuous stimulation. In response to the pulse-train stimulation, video monitoring and local field potential recording revealed associated paw movement and sensorimotor rhythms, respectively. Our results show that optogenetic stimulation of primary somatosensory cortex at beta and gamma frequencies blocks transmission of pain signals in tail clip test.

Advance in optogenetic application of cocaine addiction mechanisms / 中华行为医学与脑科学杂志

Objective To integrate existing data and analyze the optogenetic application in the study of the cocaine addiction mechanism.Methods The key words optogenetics,addiction, cocaine and so on were selected.Relevant research information was collected from various authentic databases such as China Journal Full-text Database,Chinese Biomedical Databases,Wanfang Digital Journals,VIP Full-text Database and PubMed,SCIENCEDIRECT,SPRINGERLINK databases by literature study,assess the quality of the studies and the qualitative systematic review was conducted in this study.Results Findings from present data showed that:with great spatial and temporal specificity,the optogenetic application not only proved the important role of traditional theories of mesolimbic dopamine system,but also dissected the neural circuits and molecular mechanisms of dopaminergic and glutamatergic neurotransmission underlying cocaine addiction.Conclusion The use of optogenetic tools has given researchers the potential to dissect the neuronal circuits and molecular changes,leading us to a better understanding of the mechanisms of cocaine addiction and to the development of novel treatment.

Optogenética e estimulação óptica neural: estado atual e perspectivas / Optogenetics and neural optical stimulation: current state and perspectives

Ao longo dos últimos 50 anos, o uso da luz, em especial o laser, vem promovendo grandes avanços em diversas áreas da ciência e da tecnologia. Na última década o uso de estímulos ópticos no campo da biomédica tem despertado grande interesse no meio acadêmico e na indústria. Dois ramos que se destacam pelo seu crescimento são: a estimulação óptica direta e a optogenética. A primeira utiliza diferentes parâmetros da luz para adequar o efeito desejado na interação com o tecido biológico. A segunda faz uso de engenharia genética para tornar os tecidos biológicos sensíveis à luz. A estimulação neural por infravermelho (estimulação óptica direta) não necessita de contato direto com o tecido e apresenta maior seletividade especial se comparada à estimulação elétrica, mas tem a capacidade restrita de ativar (despolarizar) os neurônios. A optogenética, entretanto, pode ser utilizada para manipular o tecido neural tornando-o sensível à luz; sendo, então, possível despolarizar ou hiperpolarizar os neurônios codificados, assim como monitorar as ativações por meio de codificação de proteínas fluorescentes sensíveis à tensão elétrica. Tanto a técnica de estimulação óptica por infravermelho ou a técnica de optogenética, vêm sendo aplicadas apenas à modelos animais. Os resultados mostram, entretanto, que há grande viabilidade de aplicação da estimulação óptica em seres humanos. Futuramente, tais técnicas poderão substituir o atual padrão ouro para a ativação neural, a estimulação elétrica, em aplicações envolvendo doenças neurológicas específicas.

Within the last 50 years the light and specially the laser has fomented great advances in several areas of science and technology. During the past decade the use of optical stimuli in the biomedical research field have been of great interest for both academy and industry. Two research branches that can be highlighted due to its growth are: direct optical stimulation and optogenetic. The first one uses different parameters of light to optimize the desired effect on the tissue interaction. The other branch works with genetic engineering technics to make cells sensitive to light. The neural stimulation by infrared (direct optical stimulation) does not require direct contact with the tissue and has higher spatial selectivity when compared to electrical stimulation, but it has restricted ability to activate (depolarize) neurons. The optogenetic, however, can be used to manipulate the neural tissue depolarizing or hyperpolarizing encoded neurons, as well as monitor activations by encoding fluorescent proteins sensitive to voltage. The stimulation by infrared optical or optogenetic, has been applied only to animal models although there is a great possibility for human applications. In the future, it may even replace existing techniques such as electrical brain stimulation to treat specific neurological diseases.