Neurite Outgrowth and Growth Cone Collapse Assays to Assess Neuronal Responses to Extracellular Cues

1989 ◽

Vol 109 (3) ◽

pp. 1229-1243 ◽

Cited By ~ 151

Author(s):

K L Lankford ◽

P C Letourneau

Keyword(s):

Neurite Outgrowth ◽

Growth Cone ◽

Actin Filaments ◽

Monovalent Cation ◽

Stabilizing Agent ◽

Growth Cone Collapse ◽

Neurite Retraction ◽

Calcium Removal ◽

The Stability ◽

Triton X

We investigated the effects of calcium removal and calcium ionophores on the behavior and ultrastructure of cultured chick dorsal root ganglia (DRG) neurons to identify possible mechanisms by which calcium might regulate neurite outgrowth. Both calcium removal and the addition of calcium ionophores A23187 or ionomycin blocked outgrowth in previously elongating neurites, although in the case of calcium ionophores, changes in growth cone shape and retraction of neurites were also observed. Treatment with calcium ionophores significantly increased growth cone calcium. The ability of the microtubule stabilizing agent taxol to block A23187-induced neurite retraction and the ability of the actin stabilizing agent phalloidin to reverse both A23187-induced growth cone collapse and neurite retraction suggested that calcium acted on the cytoskeleton. Whole mount electron micrographs revealed an apparent disruption of actin filaments in the periphery (but not filopodia) of growth cones that were exposed to calcium ionophores in medium with normal calcium concentrations. This effect was not seen in cells treated with calcium ionophores in calcium-free medium or cells treated with the monovalent cation ionophore monensin, indicating that these effects were calcium specific. Ultrastructure of Triton X-100 extracted whole mounts further indicated that both microtubules and microfilaments may be more stable or extraction resistant after treatments which lower intracellular calcium. Taken together, the data suggest that calcium may control neurite elongation at least in part by regulating actin filament stability, and support a model for neurite outgrowth involving a balance between assembly and disassembly of the cytoskeleton.

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Nogo-A Beyond the RhoA/ROCK Pathway – Novel Components of Intracellular Nogo-A Signaling Cascades

10.1101/2020.07.10.197368 ◽

2020 ◽

Author(s):

Martina A. Maibach ◽

Ester Piovesana ◽

Julia Kaiser ◽

Mea M. Holm ◽

Zorica Risic ◽

...

Keyword(s):

Neurite Outgrowth ◽

Growth Cone ◽

Signaling Cascades ◽

Growth Cone Collapse ◽

Neurite Retraction ◽

Downstream Effector ◽

Fibre Growth ◽

Growth Promoting

AbstractNogo-A is a well-characterized myelin-associated membrane protein that restricts fibre growth and the regenerative capacity of the adult central nervous system after injury. To date Nogo-A post-receptor signalling pathway research focused on the RhoA/ROCK cascade, which can lead to growth cone collapse and neurite retraction. Much less is known about continued intracellular Nogo-A signalling mediating long-term neurite outgrowth inhibition resulting from transcriptional and translational changes. Here, we propose a simple but highly reproducible in vitro assay to study Nogo-A related signaling and neurite outgrowth inhibition in general. Furthermore, we identified ERK1/2 as downstream effector of Nogo-A, partially mediating its neurite outgrowth inhibition. We describe ERK1/2 dependent changes of translational events such as elevation of RhoA levels within the growth cone, which may potentiate the cells’ responses to Nogo-A. We also observed Nogo-A dependent upregulation of the JAK/STAT pathway inhibitors SOCS3 and KLF4 and downregulation of insulin mediated phosphorylation of AKT, indicating direct negative crosstalk between Nogo-A signalling and the growth promoting JAK/STAT and AKT/mTORC1 pathways.

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Extracellular Pgk1 enhances neurite outgrowth of motoneurons through Nogo66-independent targeting of NogoA

10.1101/550921 ◽

2019 ◽

Author(s):

Cheng Yung Lin ◽

Chia Lun Wu ◽

Kok Zhi Lee ◽

You Jei Chen ◽

Po Hsiang Zhang ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Mouse Model ◽

Neurite Outgrowth ◽

Growth Cone ◽

Knockout Mice ◽

Phosphoglycerate Kinase ◽

Muscle Cells ◽

Growth Cone Collapse ◽

Nogo Receptors ◽

Lateral Sclerosis

AbstractNogoA inhibits neurite outgrowth of motoneurons (NOM) through interaction with its receptors, Nogo66/NgR. Inhibition of Nogo receptors rescues NOM, but not to the extent exhibited by NogoA-knockout mice, suggesting the presence of other pathways. We found that NogoA-overexpressing muscle cells reduced phosphoglycerate kinase 1 (Pgk1) secretion, resulting in inhibiting NOM. Apart from its glycolytic role and independent of the Nogo66 pathway, extracellular Pgk1 stimulated NOM by triggering a reduction of p-Cofilin-S3, a growth cone collapse marker, through decreasing a novel Rac1-GTP/p-Pak1-T423/p-P38-T180/p-MK2-T334/p-Limk1-S323/p-Cofilin-S3 molecular pathway. Not only did supplementary Pgk1 enhance NOM in defective cells, but injection of Pgk1 rescued denervation in muscle-specific NogoA-overexpression of zebrafish and an Amyotrophic Lateral Sclerosis mouse model, SOD1-G93A. Thus, Pgk1 secreted from muscle is detrimental to motoneuron synapse growth and maintenance.

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Extracellular Pgk1 enhances neurite outgrowth of motoneurons through Nogo66/NgR-independent targeting of NogoA

eLife ◽

10.7554/elife.49175 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 2

Author(s):

Cheng Yung Lin ◽

Chia Lun Wu ◽

Kok Zhi Lee ◽

You Jei Chen ◽

Po Hsiang Zhang ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Mouse Model ◽

Neurite Outgrowth ◽

Growth Cone ◽

Knockout Mice ◽

Phosphoglycerate Kinase ◽

Muscle Cells ◽

Growth Cone Collapse ◽

Nogo Receptors ◽

Lateral Sclerosis

NogoA inhibits neurite outgrowth of motoneurons (NOM) through interaction with its receptors, Nogo66/NgR. Inhibition of Nogo receptors rescues NOM, but not to the extent exhibited by NogoA-knockout mice, suggesting the presence of other pathways. We found that NogoA-overexpressing muscle cells reduced phosphoglycerate kinase 1 (Pgk1) secretion, resulting in inhibiting NOM. Apart from its glycolytic role and independent of the Nogo66 pathway, extracellular Pgk1 stimulated NOM by triggering a reduction of p-Cofilin-S3, a growth cone collapse marker, through decreasing a novel Rac1-GTP/p-Pak1-T423/p-P38-T180/p-MK2-T334/p-Limk1-S323/p-Cofilin-S3 molecular pathway. Not only did supplementary Pgk1 enhance NOM in defective cells, but injection of Pgk1 rescued denervation in muscle-specific NogoA-overexpression of zebrafish and an Amyotrophic Lateral Sclerosis mouse model, SOD1 G93A. Thus, Pgk1 secreted from muscle is detrimental to motoneuron neurite outgrowth and maintenance.

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