The Axon Initial Segment Plays a Dynamic Role in Peripheral Motor Neuron Synapse Regeneration following Injury
Introduction
Unlike axons of the central nervous system, which have limited ability to regenerate, axons of the peripheral nervous system (PNS) die back and can then reform in response to injury, through a process called Wallerian degeneration (Huebner and Strittmatter, 2009). However, severe peripheral nerve damage can lead to lifelong impairments including muscular atrophy, loss of function, diminished sensation, and paralysis (Navarro et al., 2007). What determines the regenerative ability of PNS neurons following injury?
The somatic nervous system is a branch of the PNS that contains two primary neuron types: sensory and motor neurons (Akinrodoye and Lui, 2022). Afferent sensory neurons communicate external information to the CNS, whereas efferent motor neurons relay CNS signals to muscles and are responsible for voluntary movement (Akinrodoye and Lui, 2022). Notably, although motor neuron axons project into the PNS, terminating on muscle fibers at a synapse known as the neuromuscular junction (NMJ), their cell bodies reside in the spinal cord, which is part of the CNS (Tintignac et al., 2015). Furthermore, motor neurons are multipolar, meaning they contain both an axon and multiple dendrites (Park et al., 2021). In contrast, the cell bodies of PNS sensory neurons are located in the dorsal root ganglion, and they are pseudounipolar, lacking dendrites and possessing a single axon that bifurcates and sends one branch to the periphery and one to the CNS (Abraira and Ginty, 2013).
Following injury, successful peripheral nerve repair requires lesioned distal axons to undergo degeneration and clearing by Schwann cells and macrophages, while axons proximal to the lesion site must retract to form a new growth cone (Shim and Ming, 2010). Reinnervation and restoration of synaptic function after injury requires synchronous upregulation of regeneration-associated genes along with growth cone-driven axon elongation. The recovery time window following peripheral nerve damage is limited, and if the repair process is not properly executed within this time frame, functional recovery and reinnervation of the target tissue may not occur (Rotshenker, 2011). Therefore, the development of therapeutics to treat peripheral axon injury requires defining the critical period for regeneration and understanding what causes the regenerative process to be disrupted.
Functional recovery of a peripheral motor neuron following injury in control mice compared with AIS-deficient mice. Top, Typically, following nerve crush injury, a neuron is able to make full functional recovery and reinnervate the NMJ by 21 DPI. This allows for proper intra-axonal trafficking and exclusion of somatodendritic particles to help maintain polarity. Bottom, In AIS-deficient mice, functional recovery and reinnervation of the NMJ is not attained by 21 DPI and proper exclusion of somatodendritic particles is diminished, contributing to the loss of polarity. Created with bioRENDER (https://www.biorender.com/).
Neuronal injury often results in structural changes or even disassembly of the axon initial segment (AIS), a 20–60 µm region of unmyelinated axon located near the cell body (Huang and Rasband, 2018). The AIS contains clusters of voltage-gated Na+ and K+ channels that are involved in action potential generation. The channels are anchored at the AIS via the major scaffolding protein AnkryinG (AnkG). AnkG is not only necessary for AIS function and stability (Hedstrom et al., 2008), but also plays a principal role in maintaining the polarity of multipolar motor neurons (Hedstrom et al., 2008). Blocking AnkG expression results in complete disassembly of the AIS, which causes axons of multipolar neurons to lose their defining structure and function (Hedstrom et al., 2008). This may be a consequence of the AIS losing its ability to exclude somatodendritic proteins, lipids, and transport vesicles from the axon (Balasanyan et al., 2017).
While the AIS has been recognized for its marked importance in neuronal polarity and action potential generation, the role of the AIS in PNS axon regeneration remains largely unexplored. Teliska et al. (2022) use a sciatic nerve crush injury model to examine the role of the AIS in peripheral axon regeneration after spinal motor neuron injury. Because AIS deficiency from birth results in lethality, the authors used transgenic mouse lines to conditionally knock out AnkG in peripheral motor axons of early adolescent mice. Intriguingly, Teliska et al. (2022) found that coordinated motor movement was unaltered in mice with AIS-deficient motor neurons compared with that of control mice, suggesting that action potential generation was intact. These results were unexpected considering the well established role of the AIS in generating action potentials (Bender and Trussell, 2009). However, this result may be explained by the clustering of voltage-gated Na+ channels found proximally at nodes of Ranvier on motor neuron axons (Huang and Rasband, 2018), which may generate action potentials in the absence of the AIS.
Axonal innervation of the NMJ was also unaltered in AIS-deficient mice, suggesting that conditional knockout of AnkG does not foster axon retraction or deterioration. After nerve crush injury, however, the time required for axon regeneration and NMJ reinnervation was significantly increased in AIS-deficient mice, suggesting a critical role of the AIS in timely regeneration of motor axons after injury (Teliska et al., 2022; Fig. 1). While these studies aimed to classify AIS function in early adolescence by studying mice aged postnatal day 21 (P21) to P30, it would be intriguing to further compare differences in regeneration from the loss of AIS function at younger and older ages, as axon regeneration following injury shows age-dependent decline (Geoffroy et al., 2017). Determining the success rate of axon regrowth in mice throughout various stages of development, and the role of the AIS in this age-dependent process, could add to our understanding of successful axon regeneration.
As mentioned above, multipolar neurons, including motor neurons, rely heavily on the AIS to maintain polarity by regulating trafficking of somatodendritic and axonal cargos (Szu-Yu Ho and Rasband, 2011). This same role is not required in pseudounipolar sensory neurons, which lack dendrites (Abraira and Ginty, 2013). Importantly, Teliska et al. (2022) found that axon regeneration in the peripherally projecting dorsal branch of pseudounipolar sensory neurons of AIS-deficient mice was indistinguishable from that of control mice following sciatic nerve crush injury. Unaltered axon regeneration in pseudounipolar neurons suggests that multipolar neurons are particularly dependent on the AIS for timely recovery following injury, perhaps because the AIS facilitates trafficking of proregeneration factors (or preventing trafficking of deleterious factors) to the injured axon (Teliska et al., 2022).
To investigate the effects of loss of AnkG on synapse function, Teliska et al. (2022) measured compound muscle action potentials (CMAPs), which reflect synchronous activation of motor neuron input and indicate synapse functionality (Ahmadian et al., 2013). Before injury, the baseline amplitude of CMAPs was lower in AIS-deficient mice than in control mice. CMAPs did not return to baseline levels in AIS-deficient mice even at 35 d postinjury (DPI), a time when structural regrowth and reinnervation of the NMJ had made a full recovery in AIS-deficient mice. Mice with an intact AIS made a full recovery to baseline CMAP levels by 21 DPI. This suggests that functional recovery was not achieved in AIS-deficient mice following injury. Motor endplates (i.e., the postsynaptic site of the NMJ) were not disrupted in this injury model, and postsynaptic acetylcholine receptors remained intact (Teliska et al., 2022). Therefore, reduced synaptic function was likely a result of impaired vesicle trafficking and/or decreased release of neurotransmitter from the presynaptic motor neuron (Teliska et al., 2022).
To understand how AIS deficiency affects neuromuscular transmission, Teliska et al. (2022) examined presynaptic terminal maturation at the NMJ using immunostaining of synaptic vesicle protein 2 (SV2), a transmembrane protein that modulates Ca2+-dependent neurotransmitter release (Madeo et al., 2014; Teliska et al., 2022). AIS-deficient mice had significantly reduced SV2 coverage at 21 DPI compared with control mice, but these levels matched those of matched controls by 35 DPI. These results suggest that AIS deficiency delays the maturation of presynaptic motor terminals. Because presynaptic release relies on multiple mechanisms beyond SV2 coverage, further examination of presynaptic vesicle trafficking, fusion, and neurotransmitter release into the synaptic cleft should be investigated to understand the mechanisms underlying decreased functional recovery up to 35 DPI in AIS-deficient mice.
Teliska et al. (2022) next explored changes in mRNA levels within the axon following the loss of AnkG. Arc, GAP43, and Nrn1, three mRNAs encoding proteins involved in axon regeneration, were reduced in AIS-deficient axons, regardless of injured state. Considering that AIS loss of function results in delayed presynaptic maturation (Teliska et al., 2022), an upstream role of the AIS in transport and localization of mRNAs may be necessary for axon regrowth.
Maintenance of neuronal polarity largely depends on distinct microtubule organization including uniform microtubule bundles passing through the AIS with the minus-ends oriented toward the soma and plus-ends toward the distal axon (van Beuningen et al., 2015). Neuronal regeneration likely requires both correct cargo entry, determined by AIS-dependent mechanisms, and proper vesicular trafficking of axonal materials, determined by correct polarization of microtubule bundles. Additional studies defining microtubule characteristics in this AIS deficiency model will be required to pinpoint these mechanisms, as the AIS cytoskeleton is rudimentary to its function (Jones and Svitkina, 2016).
Mechanisms underlying AIS selectivity and molecular transport of cargo into and out of the axon remain understudied. Gaining a comprehensive understanding of these systems could promote the development of various therapeutic targets throughout the brain and body, as the loss of polarity and impaired neuronal signaling are mechanisms highly implicated in neurodegenerative diseases such as Alzheimer's disease and dysplasia (Cid-Arregui et al., 1995; Klezovitch et al., 2004). It is likely that proper functioning of the AIS is necessary for efficient axon growth and support. Results from Teliska et al. (2022) suggest that AIS deficiency delays regeneration in multipolar motor neurons through the disruption of neuronal polarity via loss of molecular selectivity and disrupted axonal transport mechanisms. Future work will be needed to determine whether the AIS aids in regrowth primarily through retaining necessary regenerative components, such as mRNAs and proteins normally present in high levels, or an active role in promoting recruitment of these cargo. Observing local translation of mRNA at the AIS following nerve crush injury will also provide important insights into the mechanisms underlying delayed maturation in AnkG-deficient mice, as this process is implicated in synapse maintenance and regeneration (Turner-Bridger et al., 2020).
The work of Teliska et al. (2022) reveals novel functions of the AIS and paves important directions for understanding the mechanisms of axon regeneration. As the regeneration of motor neurons is not isolated to a single mechanism, future work should investigate further upstream mechanisms contributing to motor neuron regeneration. For example, endogenous dopaminergic inputs from the brain foster motor neuron generation in the ventral spinal cord of adult zebrafish (Reimer et al., 2013). Furthermore, the application of dopaminergic agonists increases motor neuron regeneration following lesions in zebrafish larva (Ohnmacht et al., 2016). Primary motor neuron signaling working in concurrence with the AIS to stimulate regeneration following peripheral nerve damage may be important for future studies aimed at promotion or acceleration of repair following injury.
Footnotes
- Received December 21, 2022.
- Revision received March 16, 2023.
- Accepted March 21, 2023.
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- Correspondence should be addressed to Joy Adler at joy.adler{at}cuanschutz.edu
- Copyright © 2023 the authors
