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Functional complexity of the axonal growth cone: a proteomic analysis.

ABSTRACT: The growth cone, the tip of the emerging neurite, plays a crucial role in establishing the wiring of the developing nervous system. We performed an extensive proteomic analysis of axonal growth cones isolated from the brains of fetal Sprague-Dawley rats. Approximately 2000 proteins were identified at ? 99% confidence level. Using informatics, including functional annotation cluster and KEGG pathway analysis, we found great diversity of proteins involved in axonal pathfinding, cytoskeletal remodeling, vesicular traffic and carbohydrate metabolism, as expected. We also found a large and complex array of proteins involved in translation, protein folding, posttranslational processing, and proteasome/ubiquitination-dependent degradation. Immunofluorescence studies performed on hippocampal neurons in culture confirmed the presence in the axonal growth cone of proteins representative of these processes. These analyses also provide evidence for rough endoplasmic reticulum and reveal a reticular structure equipped with Golgi-like functions in the axonal growth cone. Furthermore, Western blot revealed the growth cone enrichment, relative to fetal brain homogenate, of some of the proteins involved in protein synthesis, folding and catabolism. Our study provides a resource for further research and amplifies the relatively recently developed concept that the axonal growth cone is equipped with proteins capable of performing a highly diverse range of functions.

SUBMITTER: Estrada-Bernal A

PROVIDER: S-EPMC3288056 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Functional complexity of the axonal growth cone: a proteomic analysis.

Estrada-Bernal Adriana A Sanford Staci D SD Sosa Lucas J LJ Simon Glenn C GC Hansen Kirk C KC Pfenninger Karl H KH

PloS one 20120227 2

The growth cone, the tip of the emerging neurite, plays a crucial role in establishing the wiring of the developing nervous system. We performed an extensive proteomic analysis of axonal growth cones isolated from the brains of fetal Sprague-Dawley rats. Approximately 2000 proteins were identified at ≥ 99% confidence level. Using informatics, including functional annotation cluster and KEGG pathway analysis, we found great diversity of proteins involved in axonal pathfinding, cytoskeletal remode ...[more]

PMID: 22384089

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Project description:Understanding the bioenergetics of axon extension and maintenance has wide ranging implications for neurodevelopment and disease states. Glycolysis is a pathway consisting of 10 enzymes and separated into preparatory and payoff phases, the latter producing ATP. Using embryonic chicken sensory neurons, we report that glycolytic enzymes are found through the axon and the growth cone. Pharmacological inhibition of glycolysis in the presence of NGF impairs axon extension and growth cone dynamics within minutes without affecting axon maintenance. Experiments using microfluidic chambers show that the effect of inhibiting glycolysis on axon extension is local along distal axons and can be reversed by promoting mitochondrial respiration. Knockdown of GAPDH simplifies growth cone morphology and is rescued by shRNA-resistant GAPDH expression. Rescue of GAPDH using KillerRed fused to GAPDH followed by localized chromophore-assisted light inactivation of KillerRed-GAPDH in distal axons halts growth cone dynamics. Considering filament polymerization requires ATP, inhibition of glycolysis results in a paradoxical increase in axonal actin filament levels. The effect on actin filaments is because of enzymes before GAPDH, the first enzyme in the payoff phase. In the absence of NGF, inhibition of glycolysis along distal axons results in axon degeneration independent of cell death. These data indicate that the glycolytic pathway is operative in distal axons and contributes to the rate of axon extension and growth cone dynamics in the presence of NGF and that, in the absence of NGF, the axonal glycolytic pathway is required for axon maintenance.SIGNIFICANCE STATEMENT Elucidation of the sources of ATP required for axon extension and maintenance has implications for understanding the mechanism of neuronal development and diseases of the nervous system. While recent work has emphasized the importance of mitochondrial oxidative phosphorylation, the role of the glycolytic pathway in axon morphogenesis and maintenance remains minimally understood. The data reveal that the glycolytic pathway is required for normal sensory axon extension in the presence of NGF, while in the absence of NGF the glycolytic pathway is required for axon maintenance. The results have implications for the understanding of the bioenergetics of axon morphogenesis and plasticity and indicate that NGF has protective effects on sensory axon maintenance in hypoglycemic states.

Project description:BackgroundMicrotubule (MT) regulators play essential roles in multiple aspects of neural development. In vitro reconstitution assays have established that the XMAP215/Dis1/TOG family of MT regulators function as MT 'plus-end-tracking proteins' (+TIPs) that act as processive polymerases to drive MT growth in all eukaryotes, but few studies have examined their functions in vivo. In this study, we use quantitative analysis of high-resolution live imaging to examine the function of XMAP215 in embryonic Xenopus laevis neurons.ResultsHere, we show that XMAP215 is required for persistent axon outgrowth in vivo and ex vivo by preventing actomyosin-mediated axon retraction. Moreover, we discover that the effect of XMAP215 function on MT behavior depends on cell type and context. While partial knockdown leads to slower MT plus-end velocities in most cell types, it results in a surprising increase in MT plus-end velocities selective to growth cones. We investigate this further by using MT speckle microscopy to determine that differences in overall MT translocation are a major contributor of the velocity change within the growth cone. We also find that growth cone MT trajectories in the XMAP215 knockdown (KD) lack the constrained co-linearity that normally results from MT-F-actin interactions.ConclusionsCollectively, our findings reveal unexpected functions for XMAP215 in axon outgrowth and growth cone MT dynamics. Not only does XMAP215 balance actomyosin-mediated axon retraction, but it also affects growth cone MT translocation rates and MT trajectory colinearity, all of which depend on regulated linkages to F-actin. Thus, our analysis suggests that XMAP215 functions as more than a simple MT polymerase, and that in both axon and growth cone, XMAP215 contributes to the coupling between MTs and F-actin. This indicates that the function and regulation of XMAP215 may be significantly more complicated than previously appreciated, and points to the importance of future investigations of XMAP215 function during MT and F-actin interactions.

Project description:Diffuse axonal injury is a hallmark pathological consequence of non-penetrative traumatic brain injury (TBI) and yet the axonal responses to stretch injury are not fully understood at the cellular level. Here, we investigated the effects of mild (5%), very mild (0.5%) and repetitive very mild (2×0.5%) axonal stretch injury on primary cortical neurons using a recently developed compartmentalized in vitro model. We found that very mild and mild levels of stretch injury resulted in the formation of smaller growth cones at the tips of axons and a significantly higher number of collapsed structures compared to those present in uninjured cultures, when measured at both 24 h and 72 h post injury. Immunocytochemistry studies revealed that at 72 h following mild injury the axonal growth cones had a significantly higher colocalization of βIII tubulin and F-actin and higher percentage of collapsed morphology than those present following a very mild injury. Interestingly, cultures that received a second very mild stretch injury, 24 h after the first insult, had a further increased proportion of growth cone collapse and increased βIII tubulin and F-actin colocalization, compared with a single very mild injury at 72 h PI. In addition, our results demonstrated that microtubule stabilization of axons using brain penetrant Epothilone D (EpoD) (100 nM) resulted in a significant reduction in the number of fragmented axons following mild injury. Collectively, these results suggest that mild and very mild stretch injury to a very localized region of the cortical axon is able to trigger a degenerative response characterized by growth cone collapse and significant abnormal cytoskeletal rearrangement. Furthermore, repetitive very mild stretch injury significantly exacerbated this response. Results suggest that axonal degeneration following stretch injury involves destabilization of the microtubule cytoskeleton and hence treatment with EpoD reduced fragmentation. Together, these results contribute a better understanding of the pathogenesis of mild and repetitive TBI and highlight the therapeutic effect of microtubule targeted drugs on distal part of neurons using a compartmentalized culturing model.

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Functional complexity of the axonal growth cone: a proteomic analysis.

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Functional complexity of the axonal growth cone: a proteomic analysis.

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