Elsevier

Experimental Neurology

Volume 219, Issue 1, September 2009, Pages 112-125
Experimental Neurology

Local calcium-dependent mechanisms determine whether a cut axonal end assembles a retarded endbulb or competent growth cone

https://doi.org/10.1016/j.expneurol.2009.05.004Get rights and content

Abstract

The transformation of a cut axonal end into a growth cone (GC), after axotomy, is a critical event in the cascade leading to regeneration. In an earlier series of studies we analyzed the cellular cascades that transform a cut axonal end into a competent GC. We found that axotomy of cultured Aplysia neurons leads to a transient elevation of the free intracellular Ca2+ concentration ([Ca2+]i), calpain activation and localized proteolysis of submembranal spectrin. These events are associated with the formation of distinct microtubule (MT) based vesicle traps that accumulate anterogradely transported vesicles that fuse with the spectrin free plasma membrane in support of the growth process (Erez, H., Malkinson, G., Prager-Khoutorsky, M., De Zeeuw, C.I., Hoogenraad, C.C., and Spira, M.E. 2007. Formation of microtubule-based traps controls the sorting and concentration of vesicles to restricted sites of regenerating neurons after axotomy. J. Cell Biol. 176: 497ā€“507.; Erez, H., and Spira, M.E. 2008. Local self-assembly mechanisms underlie the differential transformation of the proximal and distal cut axonal ends into functional and aberrant growth cones. J. Comp. Neurol. 507: spc1.). Here we report that under conditions that limit calcium influx into the cut axonal end, axotomy leads to the formation of endbulbs (EBs) rather than to competent GCs. Under these conditions typical MT based vesicle traps are not formed, and Golgi derived vesicles concentrate at the very tip of the cut axon. Since under these conditions the spectrin barrier is not cleaved, vesicle fusion with the plasma membrane and actin polymerization are retarded and growth processes are impaired. We conclude that the immediate assembly of competent GC or an EB after axotomy is the outcome of autonomous local events that are shaped by the magnitudes of the [Ca2+]i gradients at the site of injury.

Introduction

A critical step in the regeneration of transected axons is the transformation of the cut axonal end into a specialized compartment which orchestrates in time and space the use of cell resources for growth processes (Erez et al., 2007, Erez and Spira, 2008, Sahly et al., 2006, Spira et al., 2003), and integrates extracellular signals into growth patterns, target recognition and synapse formation (for reviews Farrar and Spencer, 2008, Mortimer et al., 2008, Zheng and Poo, 2007). The failure of a cut axonal end to transform into a competent GC apparatus but rather into an endbulb (EB) or a retraction bulb was recognized as an indication for the presence of factors that interfere with regeneration by the pioneering studies of Ramon y Cajal (Defelipe and Jones, 1991). Whereas the cellular mechanisms that orchestrate the transformation of a cut axonal end into a competent GC have been investigated (Ashery et al., 1996, Erez et al., 2007, Erez and Spira, 2008, Geddis and Rehder, 2003aa,b; Gitler and Spira, 1998, Gitler and Spira, 2002, Sahly et al., 2003, Sahly et al., 2006, Spira et al., 2003, Ziv and Spira, 1995, Ziv and Spira, 1997), the mechanisms leading to the formation of an endbulb and prevent growth processes after axotomy remained elusive.

A number of factors define the ability of neurons to regrow after axotomy. Among the critical factors is the assembly of effective GC machinery. A large number of studies revealed that the formation of a competent GC depends on the microenvironment in which the axon is transected. For example, whereas lesion of the peripheral axonal branch of dorsal root ganglia (DRG) neurons leads to the formation of a competent GC and regeneration, lesion of the central branch culminates in the formation of a non-growing EB (for example Erturk et al., 2007). The differences in the response of the peripheral and central axons that extend from a common cell body are attributed to the presence of inhibitory molecules within the CNS microenvironment (Caroni et al., 1988, Caroni and Schwab, 1988 and for review Fawcett, 2006, Gonzenbach and Schwab, 2008, Hannila et al., 2007, Rossi et al., 2007). Nevertheless, since a conditioning lesion of the peripheral branch may be sufficient to alter the response of the central branch, such that it regenerates after axotomy, it was concluded that alterations of the intrinsic properties of the neuron may alter the response of a cut axonal branch to molecular cues provided by the surrounding microenvironment (for review Hannila and Filbin, 2008).

In an attempt to analyze the cellular mechanisms that lead to either the formation of a competent GC or an endbulb, the laboratory of Fawcett found that cultured DRG neurons readily form GCs when transected in normal culture medium (Chierzi et al., 2005). Nevertheless, axotomy in calcium-free growth medium ā€œimpaired the high capacity of the DRG axons to reinitiate growth coneā€. Thus, depending on the activation of molecular cascades by the incoming calcium from the cut axonal end, a transected axon either transforms into a competent growth cone or a non-growing endbulb. In a recent paper from the laboratory of Bradke analysis of DRG neurons transected in vivo and in vitro revealed that a day after lesion the retraction bulb formed at the CNS is characterized by disorganized MT network (Erturk et al., 2007). Moreover, they demonstrated that whereas MT destabilizing reagents transform competent GCs into retraction bulbs, reagents that stabilize the MTs prevent the formation of retraction bulbs. The conclusion from this study was that the organization of the MTs at the tip of transected axons, defines the fate of lesioned axonal stump to become either a competent GC or a retraction bulb (Erturk et al., 2007).

Using cultured Aplysia neurons as a model system and on line confocal imaging of cytoskeletal elements, vesicle transport and accumulation, the free intracellular calcium concentration ([Ca2+]i), calpain activity and electrophysiological methods our laboratory characterized the cascades of events that orchestrate in time and space the transformation of a cut axonal end into a competent GC (Ashery et al., 1996, Benbassat and Spira, 1993, Erez et al., 2007, Erez and Spira, 2008, Gitler and Spira, 1998, Gitler and Spira, 2002, Sahly et al., 2003, Sahly et al., 2006, Spira et al., 2003, Ziv and Spira, 1993, Ziv and Spira, 1995, Ziv and Spira, 1997). Using these studies as a basis, we examined here the mechanisms that lead to the assembly of endbulbs rather than competent GCs. We found that transection of a main neurite (referred in this paper as axon) under conditions that limit calcium influx into the cut axonal end leads through local mechanisms to the formation of endbulbs rather than competent GCs. Whereas the structural reorganization of the MTs at the cut axonal end significantly differs in axons transected under conditions that limit calcium influx from those transected under normal conditions, the formed structure provides the necessary subcellular organization to concentrate Golgi derived vesicles. Nevertheless, the fusion of anterogradely transported vesicles with the plasma membrane at the tip of the cut axon is retarded and actin assembly, to extend lamellipodia and filopodia, is greatly delayed.

Section snippets

Solutions

L-15 supplemented for marine species: Leibovitz's L-15 medium (Gibco-BRL, Paisley, Scotland) was supplemented for marine species according to Schacher and Proshansky (1983) by the addition of 12.5Ā g/l NaCl, 6.24Ā g/l D(+) dextrose, 3.15Ā g/l anhydrous MgSO4, 344Ā mg/l KCl, 192Ā mg/l NaHCO3, 5.7Ā g/l MgCl2radical dot6H2O, and 1.49Ā g/l CaCl2radical dot2H2O. Penicillin, streptomycin and amphotericin B (Biological Industries, Kibbutz Beit Haemek, Israel) were added to make final concentrations of 100Ā U/ml, 0.1Ā mg/ml, and

Formation of a competent growth cone and an endbulb by two neurites that extend from a single cell body

To examine whether Aplysia neurons form EBs when transected in low-Calcium-artificial sea water (low-Ca-ASW), we compared the outcome of axotomy of two neurites that extend from a single cell body (nĀ =Ā 25), one was transected in normal ASW (nASW, 11Ā mM Ca2+) and the other in low-Ca-ASW (0.25ā€“0.5Ā mM Ca2+). Axotomy in low-Ca-ASW was conducted as follows: 2ā€“5Ā min before axotomy the culturing medium in which the neurons were held was replaced by low-Ca-ASW. Axotomy was performed as previously

Discussion

The present study documents that axotomy of cultured Aplysia neurons, which readily regenerate a competent GC in normal ASW, forms an endbulb under experimental conditions, which limit calcium influx into the cut axonal end. On line confocal microscope imaging revealed significant differences in the [Ca2+]i gradient formed along the cut axon in nASW and in low-Ca-ASW. As a consequence, calpain is only moderately activated following axotomy in low-Ca-ASW. The differences in calcium influx and

Acknowledgments

This study was supported by grant No. 2007182 from the United Statesā€“Israel Binational Science Foundation (BSF), Jerusalem, Israel. Parts of the work were done at the Charles E. Smith Family and Prof. Elkes Laboratory for Collaborative Research in Psychobiology. We thank Dr. E. Shapira and A. Dormann for technical help in preparing mRNAs, and complementary electron microscope studies. M.E. Spira is the Levi DeViali Prof. in Neurobiology.

References (65)

  • HannilaS.S. et al.

    Therapeutic approaches to promoting axonal regeneration in the adult mammalian spinal cord

    Int. Rev. Neurobiol.

    (2007)
  • HynesR.O. et al.

    Contact and adhesive specificities in the associations, migrations, and targeting of cells and axons

    Cell

    (1992)
  • MalkinsonG. et al.

    Calcium concentration threshold and translocation kinetics of EGFP-DOC2B expressed in cultured Aplysia neurons

    Cell Calcium

    (2006)
  • McMahonA. et al.

    Calbindin-D28k buffers intracellular calcium and promotes resistance to degeneration in PC12 cells

    Brain Res. Mol. Brain Res.

    (1998)
  • MimuraF. et al.

    Myelin-associated glycoprotein inhibits microtubule assembly by a Rho-kinase-dependent mechanism

    J. Biol. Chem.

    (2006)
  • MortimerD. et al.

    Growth cone chemotaxis

    Trends Neurosci.

    (2008)
  • RoblesE. et al.

    Filopodial calcium transients regulate growth cone motility and guidance through local activation of calpain

    Neuron

    (2003)
  • RossiF. et al.

    Regulation of intrinsic neuronal properties for axon growth and regeneration

    Prog. Neurobiol.

    (2007)
  • SahlyI. et al.

    Effective expression of the green fluorescent fusion proteins in cultured Aplysia neurons

    J. Neurosci. Methods

    (2003)
  • SchoenwaelderS.M. et al.

    Bidirectional signaling between the cytoskeleton and integrins

    Curr. Opin. Cell Biol.

    (1999)
  • SchweigreiterR. et al.

    Versican V2 and the central inhibitory domain of Nogo-A inhibit neurite growth via p75NTR/NgR-independent pathways that converge at RhoA

    Mol. Cell. Neurosci.

    (2004)
  • SpiraM.E. et al.

    Use of Aplysia neurons for the study of cellular alterations and the resealing of transected axons in vitro

    J. Neurosci. Methods

    (1996)
  • BeversM.B. et al.

    Mechanistic role of calpains in postischemic neurodegeneration

    J. Cereb. Blood Flow Metab.

    (2008)
  • CaroniP. et al.

    Two membrane protein fractions from rat central myelin with inhibitory properties for neurite growth and fibroblast spreading

    J. Cell Biol.

    (1988)
  • ChierziS. et al.

    The ability of axons to regenerate their growth cones depends on axonal type and age, and is regulated by calcium, cAMP and ERK

    Eur. J. Neurosci.

    (2005)
  • ColemanM.

    Axon degeneration mechanisms: commonality amid diversity

    Nat. Rev. Neurosci.

    (2005)
  • Defelipe, J., and Jones, E.G. 1991. History of neuroscience no. 5. Cajal's degeneration and regeneration of the nervous...
  • DubreuilC.I. et al.

    Rho activation patterns after spinal cord injury and the role of activated Rho in apoptosis in the central nervous system

    J. Cell Biol.

    (2003)
  • ErezH. et al.

    Local self-assembly mechanisms underlie the differential transformation of the proximal and distal cut axonal ends into functional and aberrant growth cones

    J. Comp. Neurol.

    (2008)
  • ErezH. et al.

    Formation of microtubule-based traps controls the sorting and concentration of vesicles to restricted sites of regenerating neurons after axotomy

    J. Cell Biol.

    (2007)
  • ErturkA. et al.

    Disorganized microtubules underlie the formation of retraction bulbs and the failure of axonal regeneration

    J. Neurosci.

    (2007)
  • EvansJ.S. et al.

    Emerging functions of the calpain superfamily of cysteine proteases in neuroendocrine secretory pathways

    J. Neurochem.

    (2007)
  • Cited by (0)

    View full text