Review articleA view from the ending: Axonal dieback and regeneration following SCI
Introduction
Behavioral recovery from spinal cord injury (SCI) requires reactivation of neuronal circuitry lost as a consequence of mechanical trauma and the resulting secondary damage and Wallerian degeneration. Several experimental therapeutic approaches have been extensively examined to help preserve or enhance neuronal circuitry following SCI in an effort to restore function. These include neuroprotection [59], augmentation of sprouting [31], [33], enhancing axonal regeneration via activation of intrinsic pro-regenerative pathways [45], and reduction of environmental inhibition [18]. Significant progress has been made in promoting SCI repair in experimental models; however, even with the most promising pro-regenerative strategies, functional improvements are modest or absent, and the majority of axons still fail to regenerate past the lesion into the distal spinal cord.
With or without experimental intervention, most axons form enlarged, swollen endings following spinal cord injury [20], [47], [64], [77]. Morphologically, these enlarged endings contain disrupted cytoskeleton and accumulations of membrane and organelles [8], [25], [97]. Swollen endings form acutely near the injury site and persist for months [47] to years [82] after SCI. Given their acute formation and their persistence at the lesion margin chronically, swollen axonal endings could provide a target for both acute and chronic SCI repair if the mechanisms of their formation and persistence were better understood. Importantly, axonal endings are the location where the intrinsic growth program and extrinsic factors locally converge to execute axonal growth. Swollen endings of cut axons are frequently grouped together as a single category generically called ‘retraction bulbs.’ This likely obscures differences in these endings at different times following SCI, and hampers correct temporal targeting of therapeutic interventions.
This review focuses on the morphological characteristics and factors that impact the formation of axonal endings after injury. It explores how axonal endings change at different phases of axonal degeneration and regeneration. Factors postulated to contribute to their formation in vitro and in vivo are discussed, and areas where further research is needed are highlighted. The goal is to accentuate how axonal endings change temporally after injury; how they respond to environmental changes; and which endings current pro-regenerative therapies may act upon.
Section snippets
Axonal ending terminology
Several names have been used throughout SCI, traumatic brain injury, and peripheral nerve injury (PNI) literature in association with the proximal ends of cut axons that become swollen after injury. Within the SCI literature, the most commonly used term for the swollen endings found at or near the lesion is ‘retraction bulb.’ As retraction implies withdrawal, this name is a misnomer. The formation of these endings does not necessarily require axonal retraction, although, in some cases, their
Response of axons to injury
In order for an axon to regenerate, the axon must first stabilize the cut ending. Subsequently, it must initiate growth and then elongate. Ultimately, if regeneration is to be functionally successful, the axon must make contact with neurons and establish functional synapses. Regeneration failure can happen at any stage of this process. Contemporaneously with the attempted or thwarted growth response of the axon tip, axonal dieback can increase the distance of the axonal ending from the injury
Axonal dieback
Following injury, axon tips move away from the injury site. This is called axonal retraction, or dieback. Just how far axons die back following SCI has been a matter of debate, due in part to the use of different injury models, axon labels, and the examination of different axonal tracts. Over the last several years, a picture of how axonal endings die back from the site of injury has started to emerge.
Axonal dieback of the proximal axon occurs in several phases. Immediately following axotomy,
Axon growth failure occurs in two phases and results in different axonal ending types
In vivo imaging has provided the clearest picture of what can happen to the ends of axons following injury. In mouse dorsal column axons, 60% of axons formed an endbulb and failed to initiate growth, whereas 30% successfully formed a growth cone and subsequently achieved forward growth following axonal transection [57]. Endbulbs did not move forward following formation; however, their appearance did change during the first 48 hours [57]. Whether these changes reflect degenerative changes or
Requirements for successful growth cone formation
Initiation of axonal growth requires that the severed axon reorganize its structure and alter its function. The axon must first reseal its membrane to limit further damage. Then, it must alter its structure and change from an axon, which functioned to convey information between cells, to a dynamic growth cone, which is needed for the axon to grow in response to environmental cues. This change in phenotype requires the synthesis/recycling of proteins (such as the cytoskeletal building blocks
Endbulbs
Endbulbs form at the tips of injured axons early after injury, and accumulate organelles and membrane [54], [55]. They are, most likely, a consequence of growth cone formation failure and continued axoplasmic flow. Unlike growth cones, for which formation is dependent on calcium [15], [106] and local protein synthesis and degradation [99], the swelling of the axon and subsequent accumulation of organelles that result in endbulb formation is calcium and Ubiquitin Proteasome System
Axonal elongation
Once a growth cone has successfully formed, if regeneration is to be successful, the axon must undergo a second phase of growth: elongation. Similar to the formation of a growth cone, elongation requires altering the underlying cytoskeleton. It is likely that at least some of the signals needed for reorganization of the cytoskeleton during growth cone formation also participate in axonal elongation, as many of myelin-associated inhibitors (MAI) affect both neurite extension and growth cone
Termination of axonal elongation
Injury induces numerous cellular changes, including changes associated with axonal elongation inhibition. Axonal damage and oligodendrocyte death along with the corresponding myelin damage and degeneration leads to the exposure of MAIs, whereas changes in astrocytes and fibroblasts (along with other endogenous cellular changes) leads to the formation of the glial scar which acts as both a physical and chemical barrier to growth (reviewed in [18]). Although inhibitory components in both the
Growth cone collapse
When a growth cone collapses, it rapidly decreases in size, loses its filopodia and lamellapodia, and withdrawals slightly from the site of contact. This results from local destabilization of F-actin, which includes a loss of peripheral actin, a rapid decrease in F-actin bundles and decreased ability to polymerize new actin filaments [50]. Growth cone collapse was first termed by Raper and colleagues based on the response of different axonal types when they come in contact with one another [56]
Dystrophic axonal endings
In actively growing axons, dystrophic axonal endings form in response to gradients of inhibitory proteoglycans, but not myelin [20], [97], and they contain elevated levels of the CSPG receptors, PTPσ and LAR, but not NgRs [60]. It has yet to be established if they form in response to gradients of other chemorepellants and contact repellant molecules found after SCI (e.g. semaphorins, ephrins). In vitro, after forming, dystrophic axonal endings are initially dynamic and probe the environment in
Synapse formation
The third phase of successful axonal regeneration requires the formation of new synapses on target neurons. Contact of axons with neurons has been demonstrated for CST collateral sprouts which form en passant axonal swellings [61]. These swellings differ from the terminal swellings in that they tend to be smaller (∼3 μm), are located along the axon, and are a sign of successful innervation. Several studies have recently demonstrated synapse formation in conjunction with axonal regeneration [4],
Methodological hurdles and outstanding questions
Significant technological advances in the past decade have enhanced our ability to examine axonal endings dynamically in vitro and in vivo. Despite the progress, several challenges remain that hamper our understanding of axonal endings following SCI. Among the most pressing are the need to examine the axonal changes dynamically following injury, our limited ability to image deep within the spinal cord, and the limited number of endings that can be assessed using in vitro models.
Historically,
Conflict of interest
The author has declared that no conflict of interest exists.
Funding
This work was supported by the Burke Foundation, White Plains, NY; and the Craig H. Neilsen Foundation (Grant: 339836).
Acknowledgements
The author would like to thank: the members of the Hill lab for their assistance, in particular Ms. Taylor Johns. The author would also like to thank: Ms. Sydney Agger for artwork; and Dr. Edmund Hollis II for scientific discussions and suggestions.
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2020, Neurobiology of DiseaseCitation Excerpt :Despite much effort, the precise molecular mechanisms that cause axonal swellings, spheroid formation, and axons to retract following SCI remain poorly understood (Stirling and Stys, 2010; Hill, 2017; Ma, 2013; Bramlett and Dietrich, 2007). Among the key inducers of axonal injury, Ca2+ overload is thought to play a major role in axonal degeneration in the acute phase following SCI (Hill, 2017; Kerschensteiner et al., 2005; Stirling et al., 2014; Orem et al., 2017; Happel et al., 1981; Balentine and Spector, 1977). In support, externally-sourced Ca2+ may enter injured axons through mechanopores or nanoruptures in the axolemma (Williams et al., 2014), Ca2+ permeable receptors and channels (Li and Stys, 2000; LoPachin and Lehning, 1997; Agrawal et al., 2000), store-operated channels (Orem et al., 2020; Rao et al., 2015), and reverse operation of the Na+-Ca2+ exchanger (Bei and Smith, 2012; Stys et al., 1991).
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