RESUMO
Zebrafish exhibit robust regeneration following spinal cord injury, promoted by macrophages that control post-injury inflammation. However, the mechanistic basis of how macrophages regulate regeneration is poorly understood. To address this gap in understanding, we conducted a rapid in vivo phenotypic screen for macrophage-related genes that promote regeneration after spinal injury. We used acute injection of synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs) that were pre-screened for high activity in vivo. Pre-screening of over 350 sCrRNAs allowed us to rapidly identify highly active sCrRNAs (up to half, abbreviated as haCRs) and to effectively target 30 potentially macrophage-related genes. Disruption of 10 of these genes impaired axonal regeneration following spinal cord injury. We selected 5 genes for further analysis and generated stable mutants using haCRs. Four of these mutants (tgfb1a, tgfb3, tnfa, sparc) retained the acute haCR phenotype, validating the approach. Mechanistically, tgfb1a haCR-injected and stable mutant zebrafish fail to resolve post-injury inflammation, indicated by prolonged presence of neutrophils and increased levels of il1b expression. Inhibition of Il-1ß rescues the impaired axon regeneration in the tgfb1a mutant. Hence, our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, but can be applied to any biological function of interest.
Assuntos
RNA Guia de Cinetoplastídeos/genética , Regeneração/genética , Regeneração da Medula Espinal/genética , Fator de Crescimento Transformador beta1/genética , Proteínas de Peixe-Zebra/genética , Animais , Axônios/metabolismo , Axônios/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Modelos Animais de Doenças , Macrófagos/metabolismo , Osteonectina/genética , Recuperação de Função Fisiológica/genética , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/patologia , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/terapia , Regeneração da Medula Espinal/fisiologia , Fator de Crescimento Transformador beta3/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Being recognized as the best-tolerated of all metals, the catalytic potential of gold (Au) has thus far been hindered by the ubiquitous presence of thiols in organisms. Herein we report the development of a truly-catalytic Au-polymer composite by assembling ultrasmall Au-nanoparticles at the protein-repelling outer layer of a co-polymer scaffold via electrostatic loading. Illustrating the in vivo-compatibility of the novel catalysts, we show their capacity to uncage the anxiolytic agent fluoxetine at the central nervous system (CNS) of developing zebrafish, influencing their swim pattern. This bioorthogonal strategy has enabled -for the first time- modification of cognitive activity by releasing a neuroactive agent directly in the brain of an animal.
Assuntos
Ansiolíticos/metabolismo , Materiais Biocompatíveis/metabolismo , Sistema Nervoso Central/metabolismo , Ouro/metabolismo , Animais , Ansiolíticos/química , Materiais Biocompatíveis/química , Catálise , Sistema Nervoso Central/química , Ouro/química , Estrutura Molecular , Tamanho da Partícula , Peixe-ZebraRESUMO
Neuroscience labs benefit from reliable, easily-monitored neural responses mediated by well-studied neural pathways. Xenopus laevis tadpoles have been used as a simple vertebrate model preparation in motor control studies. Most of the neuronal pathways underlying different aspects of tadpole swimming behavior have been revealed. These include the skin mechanosensory touch and pineal eye light-sensing pathways whose activation can initiate swimming, and the cement gland pressure-sensing pathway responsible for stopping swimming. A simple transection in the hindbrain can cut off the pineal eye and cement gland pathways from the swimming circuit in the spinal cord, resulting in losses of corresponding functions. Additionally, some pharmacological experiments targeting neurotransmission can be designed to affect swimming and, fluorescence-conjugated α-bungarotoxin can be used to label nicotinic receptors at neuromuscular junctions. These experiments can be readily adapted for undergraduate neuroscience teaching labs. Possible expansions of some experiments for more sophisticated pharmacological or neurophysiological labs are also discussed.
RESUMO
Being recognized as the best-tolerated of all metals, the catalytic potential of gold (Au) has thus far been hindered by the ubiquitous presence of thiols in organisms. Herein we report the development of a truly-catalytic Au-polymer composite by assembling ultrasmall Au-nanoparticles at the protein-repelling outer layer of a co-polymer scaffold via electrostatic loading. Illustrating the in vivo-compatibility of the novel catalysts, we show their capacity to uncage the anxiolytic agent fluoxetine at the central nervous system (CNS) of developing zebrafish, influencing their swim pattern. This bioorthogonal strategy has enabled -for the first time- modification of cognitive activity by releasing a neuroactive agent directly in the brain of an animal.