By Brent Neumann
For over three millennia we have understood that injury to the spinal cord can inflict lifelong disability, with the Edwin Smith Surgical Papyrus (ca 1500 BCE) providing the earliest known diagnosis: “one having a crushed vertebra in his neck; he is unconscious of his two arms, his two legs, and he is speechless. An ailment not to be treated”. Although we have come a long way in our understanding of the nervous system since these times, it remains largely true that an injury such as that described will cause lifelong paralysis. We now have a good appreciation of the inhibitory microenvironment that precludes regeneration within the mammalian central nervous system, as well as the intrinsic mechanisms that can either promote or inhibit regrowth after injury. However, our knowledge of how target reconnection can be achieved following successful regeneration is still in its infancy.
As a postdoctoral fellow with Massimo Hilliard at the Queensland Brain Institute, I sought to address this knowledge gap by analyzing precisely how individual axons respond to injury and the mechanisms by which they can mount a regenerative response. To achieve this we exploited the experimental advantages afforded by the tiny nematode Caenorhabditis elegans (C. elegans), including the ability to visualize and sever the axons of individual neurons in living animals. Using a UV-laser to transect the axons of the C. elegans mechanosensory neurons, we identified a mechanism of repair known as axonal fusion (Neumann et al. Developmental Dynamics, 2011), a regenerative process in which severed axons spontaneously repair themselves by regrowing, reconnecting and fusing with their separated counterparts. Axonal fusion provides a highly efficient means of nervous system repair, as instead of recreating the entire axonal length beyond the site of damage, neuronal structure can be repaired by simply bridging the damaged zone. This means of repair is not limited to C. elegans, having been previously observed in several invertebrate species, including crayfish, earthworm, and leech, as well as in cultured murine neuroblastoma cells.
As a postdoctoral fellow with Massimo Hilliard at the Queensland Brain Institute, I sought to address this knowledge gap by analyzing precisely how individual axons respond to injury and the mechanisms by which they can mount a regenerative response. To achieve this we exploited the experimental advantages afforded by the tiny nematode Caenorhabditis elegans (C. elegans), including the ability to visualize and sever the axons of individual neurons in living animals. Using a UV-laser to transect the axons of the C. elegans mechanosensory neurons, we identified a mechanism of repair known as axonal fusion (Neumann et al. Developmental Dynamics, 2011), a regenerative process in which severed axons spontaneously repair themselves by regrowing, reconnecting and fusing with their separated counterparts. Axonal fusion provides a highly efficient means of nervous system repair, as instead of recreating the entire axonal length beyond the site of damage, neuronal structure can be repaired by simply bridging the damaged zone. This means of repair is not limited to C. elegans, having been previously observed in several invertebrate species, including crayfish, earthworm, and leech, as well as in cultured murine neuroblastoma cells.
Regenerative axonal fusion
Following axonal transection, axonal fusion occurs if the regenerating axonal can reconnect and fuse with its separated segment to restore membrane and cytoplasmic continuity.
Following axonal transection, axonal fusion occurs if the regenerating axonal can reconnect and fuse with its separated segment to restore membrane and cytoplasmic continuity.
For this repair strategy to be successful, there is a biological race that must be run between the regrowing axon and its separated counterpart. If regeneration is initiated too late, occurs too slowly, or takes the wrong trajectory, the internal self-destruction program within the separated axon will triumph, causing its degeneration and prohibiting its rescue via axonal fusion. As such, a sufficient delay in axonal degeneration is required for the damaged neuron to initiate its regenerative response and complete the axonal fusion process.
The biological race
Axonal injury initiates a biological race between regeneration and degeneration, which must be won by regeneration if axonal fusion is to occur.
Axonal injury initiates a biological race between regeneration and degeneration, which must be won by regeneration if axonal fusion is to occur.
Understanding the mechanisms of degeneration will ultimately allow us to manipulate the outcome of this race, ensuring that regeneration always outruns degeneration. Since 1850 when Augustus Waller first observed severed axons undergoing degeneration, it has emerged that this Wallerian degeneration is an active self-destruction process, requiring specific intrinsic molecules such as the degeneration-promoting Toll receptor adapter, Sarm1 (Osterloh et al. Science, 2012). In addition to these degeneration-inducing programs, active mechanisms function to maintain nervous system structure and prevent its degeneration. Through forward genetic screening in C. elegans, we identified the conserved α-tubulin acetyltransferase enzyme MEC-17/αTAT1 as one such maintenance molecule (Neumann & Hilliard Cell Reports, 2014). MEC-17/αTAT1 normally functions to stabilize the axonal microtubule network, with its absence leading to a disorganized network that disrupts neuronal transport and leads to the breakdown of axonal structure in an adult-onset, and progressive fashion. Interestingly, MEC-17/αTAT1 functions independently from its enzymatic function in microtubule stabilization, instead likely acting as a structural component within the microtubule lumen. Controlling the rate of axonal degeneration by promoting stabilizing molecules such as MEC-17/αTAT1, or through the inhibition of Sarm1-type self-destruction pathways could allow regeneration to triumph in its biological race against degeneration.
However, in addition to controlling the degeneration/regeneration balance, an understanding of the molecular mechanisms that mediate the axonal fusion process itself is vital for manipulating the outcome of the race. The observation of axonal fusion in the highly genetically amenable nematode provided the opportunity to characterize the process at a molecular level. Our analysis of a novel mutation that results in hyper-stable axonal microtubules revealed the importance of microtubules in the axonal fusion process, with their disruption strongly inhibiting the success rate of this repair mechanism (Kirszenblat, Neumann et al. Molecular Biology of the Cell 2013). In addition, we speculated that molecules involved in recognition and fusion events in different biological contexts may also be involved in axonal fusion, and therefore tested the ability of severed axons to undergo axonal fusion in the absence of such molecules. We discovered that regenerative axonal fusion shares much of its molecular machinery with that involved in the process of apoptosis (Neumann et al. Nature 2015). Following injury, the composition of the axonal membrane is altered, such that the phospholipid phosphatidylserine (PS), which is normally restricted to the cytoplasmic leaflet of the membrane, is flipped to the external surface to serve as a recognition, or ‘save-me’ signal for the regrowing axon. Dying cells also display this PS signal, in the form of an ‘eat-me’ signal for engulfment by surrounding phagocytes. This ‘eat-me’ signal is recognized by secreted ligands including the transthyretin TTR-52 and lipid binding molecule NRF-5 which, together with the ABC transporter CED-7, modulate the signal to promote engulfment. A critical component for the recognition process is the PS receptor PSR-1, which is expressed on the engulfing cell and directly binds the eat-me signal on the dying cell. We found that these mechanisms are largely shared with the process of axonal fusion, with the regrowing segment emulating the engulfing cell, and the separated axon analogous to the dying cell. Once reconnection between the axon segments is established, the two membranes are fused together by the actions of the fusogen molecule EFF-1.
However, in addition to controlling the degeneration/regeneration balance, an understanding of the molecular mechanisms that mediate the axonal fusion process itself is vital for manipulating the outcome of the race. The observation of axonal fusion in the highly genetically amenable nematode provided the opportunity to characterize the process at a molecular level. Our analysis of a novel mutation that results in hyper-stable axonal microtubules revealed the importance of microtubules in the axonal fusion process, with their disruption strongly inhibiting the success rate of this repair mechanism (Kirszenblat, Neumann et al. Molecular Biology of the Cell 2013). In addition, we speculated that molecules involved in recognition and fusion events in different biological contexts may also be involved in axonal fusion, and therefore tested the ability of severed axons to undergo axonal fusion in the absence of such molecules. We discovered that regenerative axonal fusion shares much of its molecular machinery with that involved in the process of apoptosis (Neumann et al. Nature 2015). Following injury, the composition of the axonal membrane is altered, such that the phospholipid phosphatidylserine (PS), which is normally restricted to the cytoplasmic leaflet of the membrane, is flipped to the external surface to serve as a recognition, or ‘save-me’ signal for the regrowing axon. Dying cells also display this PS signal, in the form of an ‘eat-me’ signal for engulfment by surrounding phagocytes. This ‘eat-me’ signal is recognized by secreted ligands including the transthyretin TTR-52 and lipid binding molecule NRF-5 which, together with the ABC transporter CED-7, modulate the signal to promote engulfment. A critical component for the recognition process is the PS receptor PSR-1, which is expressed on the engulfing cell and directly binds the eat-me signal on the dying cell. We found that these mechanisms are largely shared with the process of axonal fusion, with the regrowing segment emulating the engulfing cell, and the separated axon analogous to the dying cell. Once reconnection between the axon segments is established, the two membranes are fused together by the actions of the fusogen molecule EFF-1.
The molecular mechanisms of axonal fusion
Recognition between the regrowing axon and its separated segment occurs through similar mechanisms to those that drive apoptosis, with the severed axon exposing PS as a ‘save-me’ signal (red circles), which is bound by the secreted ligands TTR-52 (light blue) and NRF-5 (dark blue), and the membrane-bound receptor PSR-1 (gray). Bound ligands bridge the two membranes through interaction with membrane-bound receptors, which may include CED-1 (black).
Recognition between the regrowing axon and its separated segment occurs through similar mechanisms to those that drive apoptosis, with the severed axon exposing PS as a ‘save-me’ signal (red circles), which is bound by the secreted ligands TTR-52 (light blue) and NRF-5 (dark blue), and the membrane-bound receptor PSR-1 (gray). Bound ligands bridge the two membranes through interaction with membrane-bound receptors, which may include CED-1 (black).
To further drive the outcome of the race towards regeneration, a combined approach in which regenerative growth is promoted through the targeting of growth-modulating pathways may be successful. The identification of approaches that induce sustained axonal regeneration, such as the simultaneous deletion of PTEN (phosphatase and tensin homolog) and SOCS3 (suppressor of cytokine signaling 3) (Sun et al. Nature 2011) could be used to encourage robust regrowth after injury. This combined approach of promoting growth and restricting degeneration to stimulate effective regeneration through an axonal-fusion type mechanism could be used to promote more positive outcomes after nervous system injury and give consciousness back to the limbs of the injured.
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