RESUMEN
We detail the preparation of highly fluorescent quantum dots (QDs), surface-engineered with multifunctional polymer ligands that are compact and readily compatible with strain-promoted click conjugation, and the use of these nanocrystals in immunofluorescence and in vivo imaging. The ligand design combines the benefits of mixed coordination (i.e., thiol and imidazole) with zwitterion motifs, yielding sterically-stabilized QDs that present a controllable number of azide groups, for easy conjugation to biomolecules via the selective click chemistry. The polymer coating was characterized using NMR spectroscopy to extract estimates of the diffusion coefficient, hydrodynamic size, and ligand density. The azide-functionalized QDs were conjugated to anti-tropomyosin receptor kinase B antibody (α-TrkB) or to the brain-derived neurotrophic factor (BDNF). These conjugates were highly effective for labeling the tropomyosin receptor kinase B (TrkB) in pyramidal neurons within cortical tissue and for monitoring the BDNF induced activation of TrkB signaling in live neuronal cells. Finally, the polymer-coated QDs were applied for in vivo imaging of Drosophila melanogaster embryos, where the QDs remained highly fluorescent and colloidally stable, with no measurable cytotoxicity. These materials would be of great use in various imaging applications, where a small size, ease of conjugation, and great colloidal stability for in vivo studies are needed.
Asunto(s)
Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes/química , Imagen Óptica/métodos , Polímeros/química , Puntos Cuánticos/química , Animales , Azidas/química , Línea Celular , Química Clic , Drosophila melanogaster/embriología , Imidazoles/química , Ligandos , Neuronas/citología , Tamaño de la Partícula , Transducción de Señal , Compuestos de Sulfhidrilo/químicaRESUMEN
Following axotomy, a complex temporal and spatial coordination of molecular events enables regeneration of the peripheral nerve. In contrast, multiple intrinsic and extrinsic factors contribute to the general failure of axonal regeneration in the central nervous system. In this review, we examine the current understanding of differences in protein expression and post-translational modifications, activation of signaling networks, and environmental cues that may underlie the divergent regenerative capacity of central and peripheral axons. We also highlight key experimental strategies to enhance axonal regeneration via modulation of intraneuronal signaling networks and the extracellular milieu. Finally, we explore potential applications of proteomics to fill gaps in the current understanding of molecular mechanisms underlying regeneration, and to provide insight into the development of more effective approaches to promote axonal regeneration following injury to the nervous system.
Asunto(s)
Axones/metabolismo , Proteómica , Regeneración/genética , Traumatismos de la Médula Espinal/metabolismo , Axones/patología , Axotomía , Sistema Nervioso Central/crecimiento & desarrollo , Sistema Nervioso Central/lesiones , Sistema Nervioso Central/patología , Humanos , Neuronas/metabolismo , Biosíntesis de Proteínas/genética , Procesamiento Proteico-Postraduccional/genética , Transducción de Señal/genética , Médula Espinal/crecimiento & desarrollo , Médula Espinal/metabolismo , Médula Espinal/patología , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/cirugíaRESUMEN
Low-density lipoprotein receptors (LRPs) are present extensively on cells outside of the nervous system and classically exert roles in lipoprotein metabolism. It has been reported recently that LRP1 activation could phosphorylate the neurotrophin receptor TrkA in PC12 cells and increase neurite outgrowth from developing cerebellar granule cells. These intriguing findings led us to explore the hypothesis that LRP1 activation would activate canonical neurotrophic factor signaling in adult neurons and promote axonal regeneration after spinal cord injury. We now find that treatment of adult rat dorsal root ganglion neurons in vitro with LRP1 agonists (the receptor binding domain of α-2-macroglobulin or the hemopexin domain of matrix metalloproteinase 9) induces TrkC, Akt, and ERK activation; significantly increases neurite outgrowth (p < 0.01); and overcomes myelin inhibition (p < 0.05). These effects require Src family kinase activation, a classic LRP1-mediated Trk transactivator. Moreover, intrathecal infusions of LRP1 agonists significantly enhance sensory axonal sprouting and regeneration after spinal cord injury in rats compared with control-infused animals (p < 0.05). A significant role is established for lipoprotein receptors in sprouting and regeneration after CNS injury, identifying a novel class of therapeutic targets to explore for traumatic neurological disorders.
Asunto(s)
Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Regeneración Nerviosa , Receptor trkC/metabolismo , Transducción de Señal , Animales , Axones/metabolismo , Femenino , Ganglios Espinales/metabolismo , Ligandos , Neuritas/metabolismo , Plasticidad Neuronal , Neuronas/metabolismo , Ratas , Ratas Endogámicas F344 , Regeneración , Traumatismos de la Médula Espinal/patología , Activación TranscripcionalRESUMEN
Subcellular regulation of protein synthesis requires the correct localization of messenger RNAs (mRNAs) within the cell. In this study, we investigate whether the axonal localization of neuronal mRNAs is regulated by extracellular stimuli. By profiling axonal levels of 50 mRNAs detected in regenerating adult sensory axons, we show that neurotrophins can increase and decrease levels of axonal mRNAs. Neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3) regulate axonal mRNA levels and use distinct downstream signals to localize individual mRNAs. However, myelin-associated glycoprotein and semaphorin 3A regulate axonal levels of different mRNAs and elicit the opposite effect on axonal mRNA levels from those observed with neurotrophins. The axonal mRNAs accumulate at or are depleted from points of ligand stimulation along the axons. The translation product of a chimeric green fluorescent protein-beta-actin mRNA showed similar accumulation or depletion adjacent to stimuli that increase or decrease axonal levels of endogenous beta-actin mRNA. Thus, extracellular ligands can regulate protein generation within subcellular regions by specifically altering the localized levels of particular mRNAs.
Asunto(s)
Neuronas/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal , Actinas/genética , Actinas/metabolismo , Animales , Axones/metabolismo , Células Cultivadas , Proteínas Fluorescentes Verdes/genética , Hibridación in Situ , Glicoproteína Asociada a Mielina/metabolismo , Factores de Crecimiento Nervioso/fisiología , Regeneración Nerviosa , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Semaforina-3A/metabolismoRESUMEN
Neuronal cultures provide a basis for reductionist insights that rely on molecular and pharmacological manipulation. However, the inability to culture mature adult CNS neurons limits our understanding of adult neuronal physiology. Here, we report methods for culturing adult central nervous system neurons in large numbers and across multiple brain regions for extended time periods. Primary adult neuronal cultures develop polarity; they establish segregated dendritic and axonal compartments, maintain resting membrane potentials, exhibit spontaneous and evoked electrical activity, and form neural networks. Cultured adult neurons isolated from different brain regions such as the hippocampus, cortex, brainstem, and cerebellum exhibit distinct cell morphologies, growth patterns, and spontaneous firing characteristics reflective of their regions of origin. Using adult motor cortex cultures, we identify a CNS "conditioning" effect after spinal cord injury. The ability to culture adult neurons offers a valuable tool for studying basic and therapeutic science of the brain.
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Enfermedades del Sistema Nervioso Central , Neuronas , Humanos , Sistema Nervioso Central , Axones/fisiología , EncéfaloRESUMEN
The RNA-binding protein La is involved in several aspects of RNA metabolism including the translational regulation of mRNAs and processing of pre-tRNAs. Besides its well-described phosphorylation by Casein kinase 2, the La protein is also posttranslationally modified by the Small Ubiquitin-like MOdifier (SUMO), but the functional outcome of this modification has not been defined. The objective of this study was to test whether sumoylation changes the RNA-binding activity of La. Therefore, we established an in vitro sumoylation assay for recombinant human La and analyzed its RNA-binding activity by electrophoretic mobility shift assays. We identified two novel SUMO-acceptor sites within the La protein located between the RNA recognition motif 1 and 2 and we demonstrate for the first time that sumoylation facilitates the RNA-binding of La to small RNA oligonucleotides representing the oligopyrimidine tract (TOP) elements from the 5' untranslated regions (UTR) of mRNAs encoding ribosomal protein L22 and L37 and to a longer RNA element from the 5' UTR of cyclin D1 (CCND1) mRNA in vitro. Furthermore, we show by RNA immunoprecipitation experiments that a La mutant deficient in sumoylation has impaired RNA-binding activity in cells. These data suggest that modulating the RNA-binding activity of La by sumoylation has important consequences on its functionality.
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Ciclina D1/genética , Fosfoproteínas/metabolismo , ARN Mensajero/metabolismo , Proteínas Ribosómicas/genética , Regiones no Traducidas 3' , Sitios de Unión , Ensayo de Cambio de Movilidad Electroforética , Humanos , Fosfoproteínas/química , Unión Proteica , Procesamiento Proteico-Postraduccional , SumoilaciónRESUMEN
Locally generating new proteins in subcellular regions provide means to spatially and temporally modify protein content in polarized cells. Recent years have seen resurgence of the concept that axonal processes of neurons can locally synthesize proteins. Experiments from a number of groups have now shown that axonal protein synthesis helps to initiate growth, provides a means to respond to guidance cues, and generates retrograde signaling complexes. Additionally, there is increasing evidence that locally synthesized proteins provide functions beyond injury responses and growth in the mature peripheral nervous system. A key regulatory event in this translational regulation is moving the mRNA templates into the axonal compartment. Transport of mRNAs into axons is a highly regulated and specific process that requires interaction of RNA binding proteins with specific cis-elements or structures within the mRNAs. mRNAs are transported in ribonucleoprotein particles that interact with microtubule motor proteins for long-range axonal transport and likely use microfilaments for short-range movement in the axons. The mature axon is able to recruit mRNAs into translation with injury and possibly other stimuli, suggesting that mRNAs can be stored in a dormant state in the distal axon until needed. Axotomy triggers a shift in the populations of mRNAs localized to axons, indicating a dynamic regulation of the specificity of the axonal transport machinery. In this review, we discuss how axonal mRNA transport and localization are regulated to achieve specific changes in axonal RNA content in response to axonal stimuli.
Asunto(s)
Transporte Axonal/genética , Regeneración Nerviosa/genética , Proteínas del Tejido Nervioso/genética , ARN Mensajero/metabolismo , Animales , Regulación de la Expresión Génica/fisiología , Proteínas de Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Dinámicas no Lineales , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismoRESUMEN
A surprisingly large population of mRNAs has been shown to localize to sensory axons, but few RNA-binding proteins have been detected in these axons. These axonal mRNAs include several potential binding targets for the La RNA chaperone protein. La is transported into axonal processes in both culture and peripheral nerve. Interestingly, La is posttranslationally modified in sensory neurons by sumoylation. In axons, small ubiquitin-like modifying polypeptides (SUMO)-La interacts with dynein, whereas native La interacts with kinesin. Lysine 41 is required for sumoylation, and sumoylation-incompetent La(K41R) shows only anterograde transport, whereas WT La shows both anterograde and retrograde transport in axons. Thus, sumoylation of La determines the directionality of its transport within the axonal compartment, with SUMO-La likely recycling to the cell body.