15 research outputs found
Increases in Retrograde Injury Signaling Complex-Related Transcripts in Central Axons following Injury
Axons in the peripheral nervous system respond to injury by activating retrograde injury signaling (RIS) pathways, which promote local axonal protein synthesis (LPS) and neuronal regeneration. RIS is also initiated following injury of neurons in the central nervous system (CNS). However, regulation of the localization of axonal mRNA required for LPS is not well understood. We used a hippocampal explant system to probe the regulation of axonal levels of RIS-associated transcripts following axonal injury. Axonal levels of importin β1 and RanBP1 were elevated biphasically at 1 and 24 hrs after axotomy. Transcript levels for β-actin, a prototypic axonally synthesized protein, were similarly elevated. Our data suggest differential regulation of axonal transcripts. At 1 hr after injury, deployment of actinomycin revealed that RanBP1, but not importin β1, requires de novo mRNA synthesis. At 24 hrs after injury, use of importazole revealed that the second wave of increased axonal mRNA levels required importin β-mediated nuclear import. We also observed increased importin β1 axonal protein levels at 1 and 6 hrs after injury. RanBP1 levels and vimentin levels fluctuated but were unchanged at 3 and 6 hrs after injury. This study revealed temporally complex regulation of axonal transcript levels, and it has implications for understanding neuronal response to injury in the CNS
Increases in Retrograde Injury Signaling Complex-Related Transcripts in Central Axons following Injury
A comparative quantitative assessment of axonal and dendritic mRNA transport in maturing hippocampal neurons.
Translation of mRNA in axons and dendrites enables a rapid supply of proteins to specific sites of localization within the neuron. Distinct mRNA-containing cargoes, including granules and mitochondrial mRNA, are transported within neuronal projections. The distributions of these cargoes appear to change during neuronal development, but details on the dynamics of mRNA transport during these transitions remain to be elucidated. For this study, we have developed imaging and image processing methods to quantify several transport parameters that can define the dynamics of RNA transport and localization. Using these methods, we characterized the transport of mitochondrial and non-mitochondrial mRNA in differentiated axons and dendrites of cultured hippocampal neurons varying in developmental maturity. Our results suggest differences in the transport profiles of mitochondrial and non-mitochondrial mRNA, and differences in transport parameters at different time points, and between axons and dendrites. Furthermore, within the non-mitochondrial mRNA pool, we observed two distinct populations that differed in their fluorescence intensity and velocity. The net axonal velocity of the brighter pool was highest at day 7 (0.002±0.001 µm/s, mean ± SEM), raising the possibility of a presynaptic requirement for mRNA during early stages of synapse formation. In contrast, the net dendritic velocity of the brighter pool increased steadily as neurons matured, with a significant difference between day 12 (0.0013±0.0006 µm/s ) and day 4 (-0.003±0.001 µm/s) suggesting a postsynaptic role for mRNAs in more mature neurons. The dim population showed similar trends, though velocities were two orders of magnitude higher than of the bright particles. This study provides a baseline for further studies on mRNA transport, and has important implications for the regulation of neuronal plasticity during neuronal development and in response to neuronal injury
Increases in Retrograde Injury Signaling Complex-Related Transcripts in Central Axons following Injury.
Axons in the peripheral nervous system respond to injury by activating retrograde injury signaling (RIS) pathways, which promote local axonal protein synthesis (LPS) and neuronal regeneration. RIS is also initiated following injury of neurons in the central nervous system (CNS). However, regulation of the localization of axonal mRNA required for LPS is not well understood. We used a hippocampal explant system to probe the regulation of axonal levels of RIS-associated transcripts following axonal injury. Axonal levels of importin β1 and RanBP1 were elevated biphasically at 1 and 24 hrs after axotomy. Transcript levels for β-actin, a prototypic axonally synthesized protein, were similarly elevated. Our data suggest differential regulation of axonal transcripts. At 1 hr after injury, deployment of actinomycin revealed that RanBP1, but not importin β1, requires de novo mRNA synthesis. At 24 hrs after injury, use of importazole revealed that the second wave of increased axonal mRNA levels required importin β-mediated nuclear import. We also observed increased importin β1 axonal protein levels at 1 and 6 hrs after injury. RanBP1 levels and vimentin levels fluctuated but were unchanged at 3 and 6 hrs after injury. This study revealed temporally complex regulation of axonal transcript levels, and it has implications for understanding neuronal response to injury in the CNS
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Increases in Retrograde Injury Signaling Complex-Related Transcripts in Central Axons following Injury.
Axons in the peripheral nervous system respond to injury by activating retrograde injury signaling (RIS) pathways, which promote local axonal protein synthesis (LPS) and neuronal regeneration. RIS is also initiated following injury of neurons in the central nervous system (CNS). However, regulation of the localization of axonal mRNA required for LPS is not well understood. We used a hippocampal explant system to probe the regulation of axonal levels of RIS-associated transcripts following axonal injury. Axonal levels of importin β1 and RanBP1 were elevated biphasically at 1 and 24 hrs after axotomy. Transcript levels for β-actin, a prototypic axonally synthesized protein, were similarly elevated. Our data suggest differential regulation of axonal transcripts. At 1 hr after injury, deployment of actinomycin revealed that RanBP1, but not importin β1, requires de novo mRNA synthesis. At 24 hrs after injury, use of importazole revealed that the second wave of increased axonal mRNA levels required importin β-mediated nuclear import. We also observed increased importin β1 axonal protein levels at 1 and 6 hrs after injury. RanBP1 levels and vimentin levels fluctuated but were unchanged at 3 and 6 hrs after injury. This study revealed temporally complex regulation of axonal transcript levels, and it has implications for understanding neuronal response to injury in the CNS
Methods of image analysis.
<p>Time-lapse images of neurites were taken under DIC and fluorescence conditions. After classifying the neurite as an axon or dendrite using the DIC image, RNA particles (Syto, green) and mitochondrial particles (Mitotracker, red) were fluorescently labeled. (A) DIC image of a neurite (B) Both mitochondrial (B) and non-mitochondrial RNA and (C) non-mitochondrial kymographs were generated along the length of the neurite visible in the imaging field. Particles that were present in both of the kymographs were concluded to be mitochondrial mRNA whereas particles that were only present in the non-mitochondrial RNA kymograph were considered mRNA particles (arrows). (D) Overlay of mRNA kymograph (green) and mitochondrial kymograph (red). (E) mRNA kymograph without contrast enhancement. (F) Kymograph from (E) following iterative overlay of 50% transparent image to visualize dim moving particles. (G) Kymograph from (E) following contrast enhancement to visualize dim mRNA moving particles. Additional details on enhancement of dim particles are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065917#pone.0065917.s001" target="_blank">Figure S1</a>. Bar is 20 µm.</p
Characterization of fluorescent iron nanoparticles—candidates for multimodal tracking of neuronal transport
Magnetic nanoparticles were coated with either dextran or polyacrylic acid (PAA), and compared as potential traceable carriers for targeted intraneuronal therapeutics. Nanoparticles were fabricated using a chemical reduction method and their number mean diameter, aggregation, surface chemistry, crystal structure and magnetic properties were characterized. The crystalline core of the dextran-coated nanoparticles was Fe3O4, while the PAA-coated sample had an iron core. The dextran-coated iron oxide nanoparticles (DIONs) and PAA-coated iron nanoparticles (PAINs) were both stable and had a similar mean diameter of less than 10 nm. However, morphologically, the PAINs were well dispersed, while the DIONs aggregated. DIONs exhibited the presence of hysteresis and ferromagnetic properties due to aggregation, while PAINs displayed superparamagnetic behavior. Surface chemistry analysis after 2 weeks of being exposed to air indicated that DIONs oxidized to Fe2O3, while PAINs were composed of a metallic Fe core and a mixed oxidation state shell. Based on these analyses, we concluded that PAINs are stronger candidates for examining axonal transport, since they were less prone to aggregation, offered a stronger magnetic signal, and were less oxidized. Neurotoxicity analysis of PAINs revealed that no significant toxicity was observed compared to negative controls for concentrations up to 1 mg/ml, thus further indicating their potential utility for biological applications. Finally, we successfully conjugated PAINs to a fluorophore, rhodamine 110 chloride, through a simple two-step reaction, demonstrating the feasibility of functionalizing PAINs. This study suggests that PAINs should be further evaluated as a candidate technology for intraneuronal diagnostics and therapy
Hippocampal neurons are differentiated and present distinct morphology for both the axon and dendrites.
<p>Dendrites have shorter process and taper more gradually (arrows), while axons display a long, narrow process with minimal tapering (arrows in all figures). (A–C) Expression of phosphorylated neurofilaments SMI-31 (red) and microtubule-associated protein MAP2 (green) (A) Double-label immunofluorescence of SMI-31 (red) and MAP2 (green) at 4 DIV (days <i>in vitro</i>). Dendrites (MAP2) are shorter with gradual tapering projections whereas axon (SMI-31) stain display long narrow processes. (B) Double-label immunofluorescence of SMI-31 (red) and MAP2 (green) at 7 DIV. (C) Double-label immunofluorescence of SMI-31 (red) and MAP2 (green) at 12 DIV. Bar is 50 µm.</p
Summary of measured parameters used to describe transport profiles for individual particles or groups of particles.
<p>Detailed definitions are found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065917#s4" target="_blank">Methods</a> section.</p
Summary of statistically significant differences in populations and net velocities for various classes of labeled cargoes.
<p>Dash indicates no significant difference for a given parameter. Raw data are provided in supplementary figures and tables.</p