Genetics Of Wallerian Degeneration - Luke McGuinness

Genetics of Wallerian Degeneration
Luke McGuinness
20th
January 2015

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Abstract
Axonal or Wallerian degeneration occurs following a range of insults to axons both in the
central and peripheral nervous system, and is a symptom of many neurodegenerative
disorders, including Alzheimer’s disease and Parkinson’s disease. Historically believed to be
due to the passive wasting of the axon due to lack of survival factors from the cell body,
Wallerian degeneration became thought of as an active process following the discovery of a
line of mice with a genetic mutation (Wallerian Slow Degeneration, Wlds
) that could delay
degeneration significantly. While much is still not known about the mechanisms of
degeneration, this review outlines an emerging putative pathway of degeneration, and
evaluates the key players in this pathway. The elucidation of a correct model for how axonal
degeneration takes place could lead to effective therapeutic treatments for a range of
neurodegenerative diseases.
Introduction
Axonal degeneration or Wallerian degeneration was first described by August Waller in his
notable contribution to the Philosophical Transactions of the Royal Society of London. Using
frogs, he showed that the distal portion of a served nerve fibre, the portion no longer
connected to the cells body or soma, degenerates and is cleared by the immune system to
make way for new nerves to regenerate in its place (Waller, 1850). Waller’s work was built
on by Cajal, (English translation,1991), who modestly remarked in his 1914 compendium,
which provided an invaluable resource in terms of a detailed histological description of
axonal degeneration, that “I succeeded in collecting some original observations, which were
not without value”. How and why axons degenerate is a question which has stumped
researchers for some time, as understanding the mechanisms of degeneration resulting from
injury could lead to understanding of the mechanism which underlies neurodegenerative
diseases such as Parkinson’s Disease, Alzheimer’s Disease and amyotrophic lateral sclerosis
(ALS), in all of which axonal loss occurs prior to cell soma loss. Despite this, the pathway, or
more likely, pathways by which degeneration of injured axons takes place is not yet fully
understood. This review will highlight the key work which help bring our understand of axon
degeneration to where it is today, as well as commenting on some of the proposed necessary
elements for the degeneration of axons and the putative pathway in which they may lie, and
finally proposing key areas to which future research should be directed.

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Wallerian degeneration can be morphologically divided into three distinct phases (Figure 1).
Immediately after injury, there is a short period (5-30 minutes) of localised degeneration at
the section site termed acute axonal degeneration (below). After this, a latent period of up to
24 hours occurs where the axon appears normal and are still capable of conveying an action
potential (Moldovan et al., 2009). This is then followed by the third and final stage,
beginning between 24 – 48 hours after injury, where explosive degradation takes place, and
the axon fragments and is cleared away over time by the immune system, in particular
macrophages. Clearance occurs much faster in the peripheral nervous system than the central
nervous system, with this difference being due the presence of Schwann cells that help clear
myelin in the PNS (Vargas & Barres, 2007).
Figure 1: Wallerian Degeneration of Axons – Despite arising from a number of insults to the axon, the
physical degradation of the axon in most cases appears to follow the same pattern and timescale. This figure
displays degeneration initiated by section of the axon. Within minutes of injury, acute axonal degeneration
(AAD) can be seen to take place, with the ends of the axon nearest the section degenerating rapidly (Knöferle et
al., 2010). After a period of latency, at approximately 24-48 hours after injury, rapid and systemic cytoskeletal
breakdown resulting in fragmentation occurs. An increase in macrophages in both the central and peripheral
nervous systems, and an increase in Schwann cells activity in the peripheral nervous system, serves to remove
the remains of the axon and possibly promote regeneration. Diagram based on that of Wang et al., (2012).

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But why do axons degenerate following injury? The proximal site of the section site to the
cell soma begins to sprout new nerve fibres to reconstitute axon ability soon after injury
(Kerschensteiner et al., 2005), and Lunn et al. (1989) observed that lack of degeneration does
not impair regeneration in the peripheral nervous system. Why, if this is the case, do axons
choose to degrade, rather than re-joining the cell body via these new nerve shoots? Wallerian
degeneration is evolutionary conserved (Sasaki et al., 2009), insinuating an advantage in this
degradation. It may be partly explained by the fact that degeneration occurs in response to
some viral infections such as the Bunyavirus (Mukherjee et al., 2013; below), as neurons
would act as a “highway” for viruses, allowing easy access to the immune-protected brain,
and perhaps different injuries, such as section, now somehow activate these conserved
pathways of self-defence. Finding the specific causes and discovering the mechanism that
permit axonal degeneration will have important consequences for developing therapeutic
treatments to a range of neurodegenerative diseases.
Slow Wallerian Degeneration (Wlds)
A putative pathway by which Wallerian degeneration may take place, divided into initiation
and execution stages, is used during this review to add some shape to what is a highly
complex topic (Figure 4). Firstly however, it is vital to note the importance of a coincidental
discovery in 1983 of a mouse strain (C57BL/6/Ola) in which axons seemed to be protected
against Wallerian degeneration, with axons surviving significant longer than the 24hr
timespan of degeneration in their wild-type counter parts (Lunn et al., 1989). The cause of
this was eventually mapped to the 4th
mouse chromosome (Lyon et al., 1993), and was
demonstrated to be due to the presence of the slow Wallerian degeneration (Wlds
) gene, a
chimeric gene which encodes for a fusion protein (Mack et al., 2001). The Wlds
fusion
protein of 85kDa contains 70 amino acids of the N-terminal of the ubiquitin ligase UbE4b
connected to the full NMNAT1 gene by an 18 amino acid stretch (Figure 2), and its presence
in an axon conveys protection in some though not all, models of axonal degradation. The
enzymatic activity of UbE4b is not contained within this N-terminal region; it does however
contain a 16 amino acid sequence that interacts with the valosin-containing protein (VCP),
important for the localisation of Wlds
within the axoplasm and for conferring axonal
protection, as mutant Wlds
lacking this domain locates to the nucleus and does not convey
protection (Avery et al., 2009; Conforti et al., 2009). The discovery of this gene and the
protective effects which it conveys upon injured axons reignited interest in the field of the

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mechanisms behind axonal degeneration. The functions of the two elements that make up the
fusion protein in conveying protection will be discussed in greater detail below, but Wlds
is
described here as common practice in the literature evaluates the role of the presence/loss of a
particular element within the degeneration pathway by comparing the protection afforded by
their modulation, relative to that conveyed by Wlds
. As such, the description of Wlds
here
aims give the reader a frame of reference for which to evaluate the importance of the
elements discussed below to the Wallerian degeneration system.
It should also be noted that Wallerian degeneration and programmed axonal degeneration,
such as axonal pruning, occur via distinct pathways, as a number of factors which have been
shown to be necessary to the degeneration pathway, when removed, have no effect on
preventing axonal pruning (Finn et al., 2000; Freeman et al., 2012). Additionally, Wallerian
degeneration appears to be removed from apoptosis, as chemical or genetic inhibition of cell
death do not convey protection against Wallerian degeneration (Coleman, 2005). This said,
the mechanism of Wallerian degeneration is not yet fully understood, and it could be that
Wallerian degeneration and related mechanisms such as axonal pruning may converge on the
same effector steps, but have different activation pathways (Coleman, 2005).
Finally, again as is common among the literature, the phrase Wallerian degeneration used
throughout the review below refers to axonal degeneration that follows the normal time-
Figure 2: Wlds Fusion Protein - Produced by the chimeric Slow Wallerian Degeneration
(Wlds
) gene, the Wlds
fusion protein contains the full nicotinamide mononucleotide
adenylyltransferase 1 (NMNAT1) enzyme attached via a small linkage region to the 70 N-
terminal amino acids of UbE4b, an ubiquitin ligase. Notably, this UbE4b region contains the
valosin-containing protein (VCP) binding domain (N-16), essential for correct localisation
within the cell/axon. Diagram based on that of Freeman (2014).

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course of Wallerian degeneration as described above, and is sensitive to Wlds
or NMNAT
protection.
Initiation
Initial Calcium Influx & Acute Axonal Degeneration (AAD)
Historically, calcium influx at the site of injury was thought to be the signal for the activation
of Wallerian Degeneration, due to calcium’s status as an injury derived signal (George et al,
1995). This view has become outdated in recent times, as our understanding of Wallerian
Degeneration and the factors that influence it has increased dramatically. Wallerian
degeneration may be activated by many different insults to the axon, including by
interrupting the axonal transport system either genetically or by employing toxins, NMNAT2
knockdown (below) and Schwann cell protein mutation, none of which involve an initial
calcium influx (Milde et al., 2013; Samsam et al., 2003; M. S. Wang et al., 2001).
While recent studies have assigned calcium influx role later in the pathway, namely
activating the caplains, serine-threonine proteases which bring about degradation of the axon,
calcium influx has been seen to have an effect soon after injury. In earlier studies, this was
seen quite dramatically in the proximal end of the distal stump, which along with the
proximal stump to the cells soma, can be seen to degenerate rapidly after section. This was
termed acute axonal degradation and is due to Ca2+
-dependent local activation of caplain.
Caplain then acts on the proteins which maintain the integrity of the axon (below), resulting
in the breakdown of the axonal area surrounding the section. (Kerschensteiner et al., 2005;
Knöferle et al., 2010; Koch et al., 2010). However, this acute axonal degeneration (AAD)
stage is thought to be distinct from the Wallerian degeneration, though both serve to
breakdown the axon to allow its replacement (space cleared by AAD is proposed to allow a
better working environment to allow for the regeneration of the axon still attached to the cell
soma (Spira, Oren, Dormann, & Gitler, 2003) ), and both share some common steps (the
NMNAT step – Wlds
expression averts AAD) and a link if any between the two systems has
yet to be found. The AAD process is used here to illustrate the complexity of Wallerian
Degeneration, and how Wallerian degeneration might in fact be a umbrella term of a number
of different axon-death pathways with different initiators, as discussed above, but each
activating similar effector steps.

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Further studies into this area may show that AAD is a precursor step to full Wallerian
degeneration of the distal axonal fragment. However, these studies will likely prove to be
problematic, as it has been shown that inter-axonal calcium increase is needed to initiate the
future stages of Wallerian degeneration. Thus, simple chelating of calcium will not help in
elucidating this step, but instead a method of reversibly shutting of the Calcium channels
would be needed, as this would result in calcium being denied within the first few minutes of
injury only (AAD) and not affect the Wallerian degeneration stage (Wang et al., 2012).
dsarm/Sarm1
As mentioned above, Wallerian degeneration had until recently been considered a passive
process (Waller, 1850), caused by loss of communication between the cell soma and the
distal part of the sectioned axon, in addition to the interruption of the supply of key proteins
for continued cell health by the axonal transport system (termed the survival factor delivery
hypothesis (Gilley & Coleman, 2010)). While discovery of the Wlds
was vital to the field of
axon biology and to our understanding of Wallerian Degeneration, as a gain-of-function and
likely neo-morphic mutation, it did not provide conclusive evidence of an active pathway.
While a variety of genes have been shown to convey very weak protection against Wallerian
Degeneration when mutated, including IKB kinase (Gerdts at al., 2011), Dual leucine kinase
(Dlk) (Miller et al., 2009), and AKT/GSK3 (Wakatsuki, Saitoh, & Araki, 2011), it wasn’t
until the discovery of loss-of-function mutations which delay the onset of Wallerian
Degeneration to a similar extent as Wlds
that Wallerian degeneration was truly recognised as
an active process.
In a study by Freeman et al. (2012), a single mutated gene was identified in the three null
mutant lines on which axonal protection was conveyed. The axons containing this mutation
were visualised using Green Fluorescent Protein (GFP) labelling, and were seen to last up to
50 days after injury, with 100% lasting at least 30 days post section. The region containing
the nutation was named dsarm (Drosophila sterile alpha and Armadillo motif). Further
experiments, conducted using the mouse homology of dsarm, Sarm1, proved that the Toll-
like receptor (TLR) adaptor SARM1 was required for the onset of Wallerian degeneration,
and that without it, degeneration could not be initiated (Freeman et al., 2012). Sarm-/- dorsal
root ganglion neurites were also shown to protect against the withdrawal of NGF’s, though
the effect is not as lasting as that conveyed by either Wlds
fusion gene or NMNAT1
overexpression (Deckwerth & Johnson, 1994; Sasaki & Milbrandt, 2010). This may, as

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illustrated in the calcium step above, be explained by the concept of parallel systems of
axonal degeneration, which share some key steps, but are distinctive from each other in terms
of activation.
The fact that loss of Sarm1 conveys such protection upon the axon indicates that it has an
essential role to play in the pathway leading to WD, though it’s position, be it as an initiator
or an effector of the pathway, has yet to proven. However, based on current understanding, it
can be assigned to be upstream of the calcium-related activation of proteases that represent
the accumulation of the Wallerian Degeneration pathway and achieve the degeneration of the
axonal stump (Yang et al., 2013).
Additionally, due to work done on Tir-1, the C. elegans orthologue of dSarm/Sarm1, it seems
likely that Samr1 acts in response to the initial calcium influx that is associated with injury.
Normally, Sarm1 acts as a Toll-like receptor (TLR) adaptor protein, acting downstream of the
receptor and helping to propagate the signal, which is usually involved in eliciting an immune
response from the cell (O’Neill & Bowie, 2007). However, as seen in C. elegans, Sarm1
might not need to interact with a TLR in order to convey a signal. In C. elegans, and more
specifically in the context of left-right asymmetry of the worm’s olfactory receptor neurons,
Tir-1 is seen to make up part of a pathway that does not require a Toll-like receptor to be
activated. Instead Tir-1 responds to activation by Ca2+
-calmodulin kinase, and upon this,
interacts with Apoptosis signalling kinase 1 to control left-right differentiation (Chuang &
Bargmann, 2005).
The observation that Tir-1 is activated in response to a Ca2+
influx has interesting
implications for the Wallerian Degeneration pathway, as it is now being considered that
dsarm/Sarm1 may be activated by the initial calcium influx and continue to propagate the
signal throughout the rest of the axons, an idea which would answer the questions as to how
the entire axon begins to degenerate simultaneously. This was supported by the discovery that
overexpression of wild-type Sarm1 alone is not enough to begin Wallerian Degeneration,
suggesting that even when significantly over-expressed, Sarm1 requires a signal to activate
the pathway (Gerdts et al., 2013). Alternatively, some molecules produced by cells when
under attack from a pathogen can initiate cell death through TLR activation. One such
example, the interaction of microRNA let-7 with TLR7, could act as a method of signalling

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cell damage, and in this way, Wallerian Degeneration could be activated by intra-axonal
signals (Lehmann et al., 2012).
In terms of localization, investigations support the idea of Sarm1 involvement within the
Wallerian Degeneration pathway. Using epitope tagging, Sarm1 was seen to strongly localise
to the axonal mitochondria (Kim et al., 2007), which lead to the theory that Sarm1 somehow
initiates the permeability of axonal mitochondria, through formation of pores, that is
responsible for the dramatic rise in intra-axonal calcium just prior to degeneration. However
this theory was challenged when mitochondrial localisation sequences were found within
Sarm1 and subsequently deleted, and no effect on the ability of Sarm1 to promote Wallerian
Degeneration was seen (Gerdts et al., 2013).
The question as to how Sarm1 is activated, and how it then transduces the signal, remains
inconclusively answered, although the importance of the TIR (a toll-like receptor adaptor)
and SAM (sterile alpha motif) domains and their interactions have been shown to be essential
to the activation of WD (Gerdts et al., 2013). Mutation of either domain removes the
protein’s ability to activate Wallerian degeneration, while alternatively, Sarm1 mutants which
contain only the SAM and TIR domains have been shown to induce death even in uninjured
axons (Gerdts et al., 2013). This illustrates the importance of these domains, and why they
are likely targets for further research into therapeutic methods of blocking neuronal death
(below).
Finally, macrophage infiltration into Sarm-/- mutant injured nerves is reduced (see below),
indicating Sarm1 activation may sit at the beginning of a consecutive pathway that includes
degeneration, clearance of the broken down axon by the immune system and regeneration of
injured distal axons (Freeman et al., 2012).
Highwire and the Ubiquitin Proteasome System (UPS)
Behind the discovery of the Wlds
fusion gene and of dsarm/Sarm1, possibly one of the most
exciting developments in the field was the elucidation that the Drosophila E3 ubiquitin ligase
Highwire was required for Wallerian Degeneration in flies, and that loss of Highwire
conveyed protection to adult Drosophila to almost the same level of the Wlds
(Xiong et al.,
2012). This discovery evoked the idea that the Ubiquitin Proteasome System (UPS) plays a

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role in Wallerian Degeneration (Zhai, 2003) through the degradation of specific proteins that
otherwise confer protection on axons following injury.
It is interesting to note that for some time the involvement of Highwire in the activation of
Wallerian degeneration seemed counter-intuitive, as a known target of Highwire, Wallenda
(Wnd), also known as dual leucine kinase (Dlk), is a product of one of the genes mentioned
above which promotes Wallerian Degeneration in Drosophila olfactory neurons and murine
DRG (Miller et al., 2009). Surely it is counter-productive for Hiw to be degrading a protein
which has a pro-Wallerian Degeneration effect, if indeed Hiw is involved in the Wallerian
activation pathway? A partial explanation of this was found in the fact that in some neurons,
Wnd conveys a protective effect, and so is targeted for degradation (Xiong & Collins, 2012).
However, this was not true for all neuronal tissue types, and as such, the search for other
targets of Hiw began.
This search accumulated in dNMNAT, the sole orthologue of nicotinamide mononucleotide
adenylyltransferase in Drosophila, being proposed as the target for ubiquitination by
Highwire and subsequent degradation by proteasomes, based on the rapid turnover of
NMNAT2 following injury, though many factors may influence this drop in NMNAT2 levels
(below). This theory was supported by the observation that hiw mutants have increased levels
of NMNAT2 in contrast to their wild-type counterparts (Zhai, 2003).
This relative increase in NMNAT2 levels is also seen upon loss of the mouse orthologue of
Hiw, namely PAM/Highwire/Rpm1 or Phr1 (Babetto et al., 2013). Loss of Phr1 conveys
some protection upon axons, though this protection is not as strong as that achieved by Sarm-
/- mutants (Freeman et al., 2012), as Hiw axons survive for up to 10 days following injury,
compared to the minimum of 2 weeks achieved through Sarm-/- mutants. This supports the
idea that Sarm1 may be involved in activating many different areas of the pathway which run
parallel to each other, and that the activation of the UPS to degrade NMNAT2 is just one of
the avenues of Wallerian Degeneration which when employed together achieve rapid removal
and replacement of injury axons.
Despite the experiments and their finding outlined above, there is still no conclusive evidence
that dNMNAT/NMNAT2 is the target of degradation by Hiw/Phr1, and it should be noted
that while levels remain elevated in the absence of Hiw/Phr1, this does not imply a direct

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association. For example, it has been suggested that increased levels of dNMNAT/NMNAT2
could be a beneficial side effect of broadly increased protein stability due to the down-
regulation of the UPS (Freeman, 2014). It is also interesting to note that while Phr1 depletion
conveys protection on the axon, the effects are weaker than those seen by depletion of
Highwire in Drosophila (Babetto et al., 2013), possibly indicating a more complex and
integrated degeneration system in mammals.
The issue of the involvement of the UPS on a broader scale in the pathway is still under
debate. Two mechanisms have been proposed; firstly, a broad spectrum activation of E3
proteins to initiate non-specific degradation of proteins, or alternatively, an activation of
specific E3 proteins which degrade important proteins, which has a knock-on effect to begin
a second stage of degradation. The second of these mechanisms seems most likely, supports
the idea of the specific activation of Hiw/Phr1, and is well supported by the literature.
Protection of axons by proteasome inhibitors is only effective if addition is made prior to
injury, indicating an early involvement of the UPS in the Wallerian Degeneration pathway
(Zhai, 2003). However, it was also shown that microtubule fragmentation depends on the
UPS to take place, indicating that the UPS is activated later in the pathway to support caplain
in breaking down the axon (Zhai, 2003).
Finally, if the theory of specific UPS activation is correct, it raises the issue of how Hiw/Phr1
is selectively activated. Following the rule of thumb that often the simplest explanation is the
correct one, could an interaction of dsarm/Sarm1 and Hiw/Phr1 results in the degradation of
dNMNAT/NMNAT2? Alternatively, is there an intermediate element that propagates the
signal from one to the other that thus far has remained elusive? A lack of research into this
particular area is evident from the literature, though elucidation of this step would greatly
increase current understanding of the Wallerian degeneration pathway.
Nicotinamide mononucleotide adenylyltransferase (NMNAT)
The importance of nicotinamide mononucleotide adenylyltransferase (NMNAT) within the
Wallerian degeneration pathway has been frequently mentioned, with Hiw selectively
targeting this enzyme for degradation. The importance of NMNAT presence for continued
cell health was deduced from the discovery of the Wlds
fusion gene. Responsible for the
conversion of nicotinamide mononucleotide (NMN) to form nicotinamide adenine
dinucleotide (NAD+
), NMNAT allows for the cellular respiration. Based on their localization

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and functional differences, three human NMNAT isoforms are recognised under normal
conditions (Berger et al., 2005), with a tentative fourth isoform being proposed in the form of
Wlds
fusion protein (Orsomando et al., 2012). NMNAT1 is located in the nucleus, NMNAT2
is located in the cytosol (Figure 3), while Nnmat3 is thought to associate with the
mitochondria, and is the main producer of NAD+
for respiration (Garavaglia et al., 2002;
Raffaelli et al., 2002; Yalowitz et al., 2004). The three enzymes are supposed to have
different functions beyond the production of NAD+
as NMNAT2 deletion cannot be rescued
by NMNAT1/NMNAT3 (Gilley & Coleman, 2010). NMNAT2 is also transported from the
nucleus where it is produced via the axonal transport system, a system of tightly controlled
Golgi-derived vesicles transported by the motor protein kinesin, which ensures adequate
levels of NMNAT2 along the length of the axon.
The loss of NMNAT2 could be considered to be an early convergence point in the Wallerian
Degeneration pathway. The subject of many papers regarding how it regulates axon survival
and growth, studies based on mouse embryos demonstrate the depletion of NMNAT2 results
in defective axonal growth, resulting in embryonic lethality (Gilley et al., 2013; Hicks et al.,
2012). Additionally, NMNAT2 is essential to continued axon health after injury, as identified
by a 2010 study which showed that endogenous NMNAT2 is a survival factor for axons, and
that its replenishment from the nucleus by the axonal transport system to the rest of the axon
is a key Wallerian Degeneration delaying factor. It demonstrated this by showing for the first
time that NMNAT2 is present in neurites, projections from a nerve cell body, and that level
of NMNAT2 decrease in the distal end of the axon to the injury/section prior to the onset of
degeneration. Additionally, the introduction of sufficient amounts of exogenous NMNAT2
was enough to convey a protective effect on the severed axon, demonstrating that for
Wallerian degeneration to take place, the levels of NMNAT2 within the distal stump must
fall below a certain threshold (Gilley & Coleman, 2010a). Finally, NMNAT2 is transported
through the cell via a Golgi-derived axonal transport vesicle, and association of NMNAT2
with the vesicles is achieved though palmitoylation of a dual-cysteine motif, found within the
isoform-specific targeting and interactive domains (ISTID) (Lau et al., 2010). Deletion of this
site leads to dissociation of NMNAT2 from the vesicle, and increases its potency as a
protector against Wallerian Degeneration (Milde, Fox, et al., 2013). This is in part due to the
reduced ubiquitination and subsequent degradation that takes place due to NMNAT2 no
longer being localised to the axonal transport vesicles (Milde, Gilley, et al., 2013).

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Figure 3: Location of nicotinamide mononucleotide adenylyltransferase (NMNAT)
isoforms within the neuron – NMNAT1 is primarily located within the nucleus, NMNAT2 is
has a cytosolic/axoplamsic localisation, while NMNAT3 is believed to localise to the
mitochondria and to be the main producer of the nicotinamide adenine dinucleotide (NAD+
)
required for oxidative phosphorylation and the production of ATP. The location of action of
the Wlds
fusion in conveying protection has not yet been conclusively proven, and this is
reflected in the diagram above, with Wlds
being shown to be present in the axoplasm and
nucleus, as well as co-localising with the axonal mitochondria. Diagram based on that of
Conforti et al. (2014).
The importance of NMNAT family in the Wallerian degeneration pathway was originally
considered to be in terms of NAD+
and subsequent ATP production, as without energy the
cell cannot survive. NAD+
levels are seen to drop before degeneration, and this drop is not
due to leakage from the injury site, as a study showed that other small molecules, for
example nicotinic acid, did not decrease (J. Wang et al., 2005). This drop in NAD+
levels was
thought to have a role in activation of Sirt1 (below) and of a calcium influx. However a
recent paper show that while exogenous introduction of NMNAT1, the full gene of which is
included in the Wlds
chimeric gene, into the axon prevents Wallerian Degeneration, increase

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levels of NAD+
through either genetic or pharmacological means does not convey protection
onto the injured axon (Sasakiwt al., 2009),. The paper on this study proposed that a currently
unknown enzymatic function of NMNAT2 is what is responsible for the delayed onset of
Wallerian Degeneration, and not the production of NAD+
. However, this is at odds with a
second study which showed that application exogenous NAD+
, which can be transported
across the membrane, was enough to convey protection on the axon, and suggested that the
NMNAT protection was a result of the local effect on cellular bioenergetics that the
continued production of NAD+
has (J. Wang et al., 2005). The disparity between the studies
above and more in this particular area of the pathway, which often produce very variable
results, may be due to the methodology employed, as often studies look at the application of
non-physiological amounts of NAD+
, or find no protection is afforded at all (Araki, Sasaki, &
Milbrandt, 2004; Sasaki, Araki, & Milbrandt, 2006; J. Wang et al., 2005), results which
influence the thinking around the effect this particular substrate has on the system.
However, it is vital to consider the importance of one of the other isoforms of NMNAT,
namely NMNAT1, to the Wallerian degeneration pathway, as its role in protection via the
Wlds
chimeric fusion gene. NMNAT1 is predominantly expressed in the nucleus, due to its
nuclear location signal (NLS), but deletion of this signal creates a mutant form of NMNAT1
found in the cytoplasm, namely NMNAT1cyto
, and supresses degeneration to a level that is
comparable to Wlds
. Addition of an axonal localisation signal to this NMNAT1cyto
mutant
improves its ability to protect the axon dramatically (Babetto et al., 2010). This was
supported by a study showing that mice with NMNAT1 and a level of NAD+
production on
the same level as Wlds
had no axonal protection following injury. This would indicate that
the localisation of the three isoforms is partially responsible for their non-redundancy, and
that NMNAT1 is also capable of executing the same unknown enzymatic function as
NMNAT2, which propagates the Wallerian degeneration. This is interesting, as it challenges
the assumption that Wlds
protection via NMNAT1 activity was nuclear-based. NMNAT1
enzymatic function, and demonstrates that overexpression is not enough to convey protection
to the axon. (Conforti et al., 2009). Further experiment supported this thinking, and swayed
the idea of NAD+
regulating the transcription of survival genes through histone de-acteylase
Sirtuin 1 activity (below) (Araki et al., 2004). The part of the fusion protein responsible for
localising a small part of the overall cellular Wlds
content in mice containing the fusion gene
was located to the furthermost 16 amino acids of the N-terminal region, where the valosin-
containing protein domain is located (Laser et al., 2006). The importance of this “N16

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region” to the protective effect of Wlds
was discovered though a number of experiments.
Deletion of the region resulted in lack of protection in Drosophila and mice tests, while
knocking down of VCP in Wlds
models in Drosophila reduced its protective effect to that of
NMNAT1 only (Avery et al., 2009; Conforti et al., 2009). These experiments are outlined
here to demonstrate that NMNAT function for continued axonal survival and axonal
protection in the Wlds
is due to some enzymatic function in the cytoplasm/axoplasm, and not
in the nucleus as originally thought.
Further narrowing down the area within the cytoplasm/axoplasm which NMNAT could
function to convey axonal protection, upon deletion of its NLS, the Wlds
protein has been
shown to give extreme, above normal Wlds
protection against degeneration and to localise to
the mitochondria (Beirowski et al., 2009), a fact which may hint at a function in axonal
mitochondria. In keeping with this idea mouse test and Drosophila test, NMNAT expression
in the mitochondrial membrane was seen to convey protection to a Wlds
level (Yahata et al.,
2009), while studies examining the mitochondria of Wlds
mice have reported an increased
ability to produce ATP, to buffer Ca2+
, and to prevent formation of permeability transition
pores (PTP), which form on the mitochondrial membrane (Avery et al., 2012; Yahata et al.,
2009). Each of these observations points towards a mitochondrial impact for NMNAT, and
are each important for continued cell survival. Intriguing in particular is the increased ability
to buffer extra mitochondrial Ca2+
, as the proposed final step in the Wallerian degeneration
pathway is a calcium-dependent process. Interesting also is the decrease of reactive oxygen
species ROS production in Wlds
mitochondria, as ROS production usually pre-empts
Wallerian degeneration
It can be seen from the above that the current understanding of the system by which the
NMNAT family convey protection is not fully understood, and that consensus does not seem
forthcoming. As shown above, loss of NAD+
could be viewed as an important effector of the
degeneration pathway, and based on current research, loss of NMNAT2 is understood to be
the most important reversible event within the pathway on which a multitude of proposed
activating steps may converge. It is important to consider that the correct pathway might
involve the application of both ideas expressed above. Axonal protection is not conveyed by
enzymatically dead Wlds
in mice (Conforti et al., 2009), but is this due to the production of
NAD+
or some other function of the NMNAT protein? Could depletion of NAD+
be part of
the same pathway as NMNAT’s mysterious secondary enzymatic function? Could this

15
pivotal step, on which many different pathways are thought to converge, act like a bottle
neck, itself activating a host of downstream parallel pathways which work in unison to
achieve degeneration? From the above, loss of NMNAT2 could impact ATP production,
mitochondrial membrane stability and the production of ROSs. The importance of these areas
to Wallerian degeneration pathway is considered in more detail below.
Execution
Sirtuin 1
Historically, NMNAT was thought to convey its axonal protection through the activation of
Sirtuin 1(Sirt1), a NAD+
-metabolising deacetlyase. Sirt1 , upon activation was proposed to
modify histones in order to control the expression of protective genes (Araki et al., 2004). A
2004 study provided siRNA evidence for the involvement of Sirt1, which is a member of the
family of homolog proteins to the Sir2 protein found in S. cerevisiae, and studies before this
demonstrated that Sirt1 activity could down-regulate the p53 tumour suppressor gene as a
stress response in mammals to control whether the cell survived (Vaziri et al., 2001). Since
that time however, the amount of evidence against the involvement of Sirt1 in the regulation
of NMNAT2 protective ability has grown dramatically. Cultured neurons do not have raised
levels of NAD+
when overexpressing NMNAT1 or Wlds
(Araki et al., 2004), calling into
question how Sirt1 was upregulated following injury. Additionally, in sirt1knockout mutants,
NMNAT1 and Wlds
protection remained in DRG cultures (J. Wang et al., 2005), again
suggesting that Sirt1 was not involved in providing axonal protection, and as such, the Sirt1
gene would not affect the Wallerian degeneration. Instead, the effectors responsible for
activation of the final step, the physical degeneration of the axon, are proposed to be calcium
influx and the creation of reactive oxygen species.
Calcium
The need for a rise in inter-axonal calcium in order for Wallerian degeneration to progress
was demonstrated through chelating extra-axonal Ca2+
with EGTA, which protects the axon,
delaying degeneration by up to 4 days (Schlaepfer & Bunge, 1973). This loss-of-function
experiment was also supported by another study, which showed that addition of calcium
ionophores to healthy axons could induce degeneration (George et al., 1995). Additionally,
we can place calcium-dependent degradation as downstream effector of the Wallerian
degeneration pathway due to a study which shows that addition of Ca+
is capable of
overriding the protection effects of the Wlds
-fusion gene, demonstrating that calcium-

16
dependent degradation is downstream of the process which mediate the activation of
Wallerian Degeneration (Glass et al., 1994).
Looking at Wallerian degeneration from a broad viewpoint, there are three main methods by
which this required intra-axonal calcium rise can be achieved. Initially, a small influx of
calcium can occur through the injury site, though this influx is quickly restricted by the
sealing of the membrane at the section site by the Ca2+
-dependent fusion of vesicles
(Eddleman et al., 1998). Also, if this small Ca2+
influx was to be enough to induce
degeneration, it raises the question as to why the proximal side of the section, the part of the
axon still attached to the cell soma, does not undergo similar degeneration to the distal part.
Some local degeneration is seen in the form of AAD (above), but nothing on the same scale
of the total axonal disintegration of Wallerian degeneration is seen. This indicates that while
this calcium influx may be permissive, a secondary signal is needed for the activation of
degeneration – could this perhaps be the loss of NMNAT2 observed above? Finally, the
physical process of Wallerian degeneration of the axon occurs some time (~24 hours) after
injury is sustained, indicating that the initial calcium influx is not enough to immediately
activate caplain, and increased calcium levels can been seen throughout the axon, especially
in the distal end of the severed axon to the section (Ziv & Spira, 1993), again indicating an
alternative mechanism by which they are achieved.
While NAD+
was shown above to not be responsible for regulating the Wallerian
Degeneration pathway by means of Sirt1, it may still play a role in terms of the fact that loss
of NMNAT activity/NAD+
leads to ATP depletion. NAD+
acts as an electron donor within
the electron transport chain, and as such is vital for ATP production within the cell. Levels of
ATP within injured axons have been shown to drop prior to degeneration (J. Wang et al.,
2005), and it is proposed that this drop in the cells energy levels has a profound effect on the
axonal membrane and is key in initiating the Ca-influx associated with Wallerian
degeneration. One proposed mechanism is that failure of the Na+
/K+
ATPase channels, due to
the reduced levels of ATP allows a nominal influx of Na2+
into the cell through Tetrodotoxin-
sensitive Na2+
channels. This Na2+
influx was cited as the cause of a membrane
depolarisation, and allowed a Ca2+
influx via voltage gated Ca2+
channels (Stirling & Stys,
2010). This would explain how the observation above that calcium levels increase throughout
the sectioned axon, as ATP loss is systemic. However, when these Na2+
channels were
blocked using tetrodotoxin, no protection was conveyed on the axon (George et al., 1995),

17
diminishing the importance of the Na+
influx. However, decreases in K+
levels within the cell
have been shown to have a role in degeneration, as a decrease in K+
levels within the cell
occurs prior to Na+
/Ca2+
increase (Mishra et al., 2013), suggesting that influx of Na+
may still
take place and induce calcium increase, but through a different mechanism involving an
initial loss of K+
from the cell. In conjunction, these findings support a role for K+
/Na+
in
controlling the permeability of the axonal membrane to calcium, the second proposed source
of intra-cellular calcium. This need for NAD+
levels within the cell is also supported by other
studies. In Wlds
mice, properly functioning mitochondria were seen to be essential for
protection, as upon the addition of carbonyl cyanide m-chlorophenyl-hydrazone, a chemical
which uncouples oxidative phosphorylation, Wlds
protection is no longer seen (Ikegami &
Koike, 2003). This suggests an important role for NAD+
depletion and loss of functioning
mitochondria as key points in the degeneration pathway.
However, in contrast to this, alternative experiments play down the impact of NAD+
levels in
propagating the degeneration signal. One study showed that overexpression of NMNAT3
within the mitochondria delayed axonal degeneration (Press & Milbrandt, 2008), as well as
demonstrating that protection was conveyed even in the presence of Rotenone, an inhibitor of
oxidative phosphorylation, indicating a diminished role for the drop in local energy levels in
the degeneration pathway. Additionally, in vitro inhibition of Nampt, an enzyme which
produces NMN, the precursor to NAD+
, did not remove protection afforded by Wlds
, while in
vivo, a small inhibition of Wlds
protection was seen in Drosophila, but could be due to other
factors, such as the toxicity of the Nampt inhibitor to the system. These studies together
increase the weight of evidence that points place the loss of the as yet undiscovered
secondary enzymatic function of the NMNAT family of proteins as the key driver of
degeneration, above the loss of NAD+
.
The third and final proposed source of calcium influx, beyond that achieved from the injury
site and through NAD+
loss, examines the role of intra-cellular Ca2+
stores, such as the axonal
mitochondria and ER/Golgi, in terms of moderating the calcium levels within the axon.
Substantial amounts of calcium are stored in these structures (Verkhratsky, 2005), and by
tightly coordinating influx/efflux, these organelles maintain homeostatic intra-cellular
calcium levels. In terms of controlling the flow of calcium between their interior and the
axoplasm, mitochondria are able to release Ca2+
via permeability transition pores (PTP) in
response to high internal mitochondrial calcium levels (Rasola & Bernardi, 2007), the

18
formation of which Wlds
murine mitochondria are shown to be resistant to (Avery et al.,
2012). Mitochondria also sport selective uniporters, 40 kDa proteins located on the
mitochondrial inner membrane (De Stefani et al., 2011; Patron et al., 2013), which can be
employed in the uptake of calcium from the axoplasm. The axoplasmic reticulum, the axonal
counterpart of the endoplasmic reticulum (Lindsey & Ellisman, 1985; Merianda et al., 2009) ,
also plays a part in the regulation of intra-cellular calcium levels, and evidence of an
interaction between the two organelles at regions of high calcium concentration to maintain
normal levels demonstrates that a dynamic system of calcium control is at work (Rizzuto et
al., 1998).
But how do these organelles contribute to the massive increase in calcium levels seen in the
cell prior of degeneration? It was originally proposed that the initial influx of calcium form
the injury site, along with the nominal influx gained from the loss of ATP as outlined above,
was enough to overwhelm the buffering capacity of the axon and cause a mass efflux from
intra-cellular storage site, leading to caplain activation. However, as injury is not the only
method of causing Wallerian Degeneration (above), calcium influx cannot be the only way in
which the PTP are formed on the mitochondria, allowing efflux. Studies have noted that
NAD+
is able to moderate ion channels (Tamsett et al., 2009), leading to the proposal that
loss of NMNAT activity, and subsequently of NAD+
, may lead to an increase in intracellular
calcium release from these storage sites.
Sarm1
Finally, overexpressed GFP-tagged murine Sarm1 has been seen to associate with neuronal
mitochondria (Gerdts et al., 2013), and it is thought that it may play some role in the
formation of PTPs and the creation of a calcium increase. However, when the mitochondrial
location site with Sarm1was discovered (Panneerselvam et al., 2012), and a genetic mutant of
Sarm1 created without this sequence (Gerdts et al., 2013), Sarm1 was seen to be independent
of the mitochondria, and no protection of axons was seen. Additionally, endogenous Sarm1 is
seen to only tentatively localise to the mitochondria compared to overexpressed Sarm1
(Gerdts et al., 2013), indicating that this association might be the result of overexpression
rather than Samr1’s true location. These findings together demonstrate that the role of Sarm1
is largely cytosolic, such as the activation of Highwire (above), and that localisation to the
mitochondria is not essential for its function in the pathway. As a closing remark on the
interaction of Sarm1 with the mitochondria, it is intriguing, as observed by Mukherjee et al.

19
(2013), that Sarm1 relocalises to the mitochondria in response to infection by the Bunyavirus
pathogen, which involves mitochondrial damage in its method of neuronal death, an event
which could prove to occur as part of the Wallerian Degeneration pathway.
It is again important to take the broader view, and to consider the system of Wallerian
Degeneration as a whole host of pathways, sharing some key steps, which work together to
achieve degeneration. This is especially true of the execution pathway, for, as cane be seen
above, the number of possible knock-on effects that the loss of NMNAT2 may have within
the cell are many.
Regarding the physical degradation of the axon, current opinion is much more aligned on
how this particular step is achieved. The calcium influx that is observed just prior to
degeneration (George et al., 1995) activates Ca2+
-dependent caplains. The importance of
caplains has long been recognised as the main effector of the Wallerian Degeneration
pathway, as through their protease function they are responsible for the cleavage of
components of the axonal cytoskeleton such as spectrin, which plays a role in cytoskeleton
structure maintenance and plasma membrane integrity, and tubulin, a microtubule associated
protein (Billger et al., 1988; Huh et al, 2001; Johnson et al, 1991). The proteolytic activity of
caplains is what was believed to be the cause of degeneration, and a 2013 study proved that
caplains were indeed required by expressing human calpastatin (hCAST) in murine sciatic
axons. Calpastatin is an inhibitor of caplain activity, and following nerve section, protection
of the axonal cytoskeleton could be seen up to 48 hours after injury, demostrating that
caplains were responsible for the morphological degradation of the axon (Ma et al., 2013).
However, while caplain inhibition by pharmacological means prevents cytoskeletal
degradation in injured DRG neurites, it is not capable of preventing blebbing, possibly due to
unsuccessful protection of the microtubules, the degradation of which was shown to involve
the UPS (Zhai, 2003). Interestingly, calcium chelation experiments, similar to those described
above, were far more successful in conveying axon protection than those inhibiting the
activity of caplains, indicating that Ca2+
has a secondary pathway which it activates to effect
degeneration (Zhai, 2003).
A secondary system by which degeneration is thought to be achieved is through the creation
of reactive oxygen species (ROS). Molecular oxygen (O2) acts as a terminal electron receptor
as it can be reduced to stable water during oxidative phosphorylation. This is achieved

20
through Complex III of the electron transport chain, located in the mitochondria membrane,
and under normal circumstances, this reduction is achieved with the creation of a minute
amount of reactive intermediates (Davies, 1995). However, it is possible that as NAD+
levels
within the cell drop and mitochondrial membrane is lost, an increase in the creation of ROS
may occur as well. ROSs are dangerous to the cell, as they can oxidise macromolecules
within the cytoplasm and damage the cell’s DNA, the former of these two modes of action
being particularly relevant to the degeneration pathway, and ultimately lead to axonal
degeneration (Fucci et al., 1983). The importance of the creation of ROS to the Wallerian
Degeneration pathway was shown through the fact that ROS production increase in zebrafish
just prior to degeneration, and that Wlds
mutants show increased resistance to the formation
of ROSs (O’Donnell et al., 2013), though whether this resistance is a by-product of the true
protective mode of action of the fusion protein, for example increasing the level of NAD+
in
the axon or inducing a Ca2+
buffering system, has yet to be conclusively proven.
However, the role of the mitochondria, which based on the evidence above could be said to
be quite important in achieving degeneration, is by no means uncontested. A recent study
showed that, despite genetically silencing the mitochondria in Drosophila motor neurons,
Wallerian degeneration still took place, and that NMNAT/ Wlds
protection could be
conveyed despite the lack of mitochondria in the axon (Kitay et al., 2013).
Other mediators
Despite the putative pathway laid out above, it is vital to remember that there is a lot more to
be done before we can conclusively assign initiators and effects to the Wallerian pathway,
and beyond that, assign where in the pathway they act to propagate the signal. Many enzymes
that have been shown to impact Wallerian degeneration were excluded from the above for
reasons of clarity, some of which are described here.
Inhibitor of KB kinase (KKB) has been shown to have a pro-degenerative effect in DRG
cultures across injury and non-injury models, while the fact that inhibition of these kinases
only convey protection when applied at the time of injury, indicating an early acting role, if
any, for this mediator (Gerdts et al., 2011). However, the protection achieved genetic
mutation of the gene encoding for this protein (above) is weak relative to that conveyed by

21
Wlds
(Gerdts et al., 2011), suggesting that while it has been shown to moderate the
degeneration pathway, the extent to which it does is not, at present, recognised to be
significant.
Additionally, the role of serine protease within the pathway has yet to be assigned. Inhibition
of serine protease activity has been shown to convey protection in a number of studies.
Similarly to IKK, inhibitors only convey protection if added around the time of injury, again
indicating an early acting role. Again, protection acquired from loss of serine protease
function is not as strong as that afforded by Wlds
, indicating that the proteases are involved in
the pathway in a mediating role.
Finally, and possible most interestingly, the role of SG10 within the pathway has yet to be
conclusively decided upon. A labile protein, loss of SCG10 occurs early in the pathway, and
is thought to act as a permissive signal for the onset of Wallerian degeneration. Intriguingly,
SCG10 degradation is linked to the JNK (JUN amino-terminal kniase) cascade, which
involves Wallenda, or Dual leucine kinase in mammals (Miller et al., 2009), a degeneration
permissive factor which raised discussion when it was seen to be counterintuitively degraded
by Highwire/Phr1 as part of the degeneration pathway (above). However, in the future, it will
be interesting to see if the fact that SCG10 appears to localise to the Golgi derived vesicles
through palmitoylation, which is similar to NMNAT’s behaviour in the axon, implies an
interaction between the two.
Clearance
The final step of the degeneration pathway that needs to be considered is the clearance of the
myelin sheath following fragmentation of the axon as a result of the activation of caspases
and production of ROS (above). Soon after degeneration, Schwann cells in the peripheral
nervous system react to axonal loss by down-regulating myelin protein synthesis (LeBlanc &
Poduslo, 1990), fragmenting their myelin sheaths and dividing to form bands of Bunger,
serving as a guidance system of sorts for the regenerating nerves from the stump proximal to
the cell soma (Stoll et al., 1989). Additionally, macrophage recruitment is required to aid the
Schwann cells in clearing remnants of the in the PNS, following fragmentation and
cytoskeletal breakdown of the degenerating axon. Sarm1 has been shown to have a role in
producing cytokines to attract macrophages to help in the clearance of the myelin sheath, a.

22
Figure 4: Putative Wallerian Degeneration Pathway – The main elements of degeneration
described in the main text above have been assembled here into a putative pathway, drawing from
that proposed by Conforti et al, 2014. This pathway focuses predominantly on degeneration caused
by physical injury to the axon, for example section, and as such other proven initiators of
Wallerian degeneration of axons, such as toxins or NGF withdrawal (see main text), have been
omitted. The axoplasmic reticulum, a second source of intra axonal calcium has also been omitted
for clarity. Calcium is shown above as both an initiator, in terms of the proposed activation of
Sarm1, and as an effector through the activation of caplains which degrade cytoskeletal and
microtubule associated compound. A solid line indicates an interaction, while dotted line
represents a pathway that has yet to be experimentally proven, but which is likely to take place
either directly or through a intermediate, such as the interaction between Sarm1 and Phr1. It
should also be noted that there are gaps within the above pathway, and as the field progresses, it is
inevitable that more as yet unknown elements of the degeneration pathway will emerge. The above
pathway was created using PathVisio (van Iersel et al., 2008).

23
finding which might have been expected as Sarm1 is a member of the toll-like receptor
adaptor protein family, known to be involved in innate immune response (O’Neill & Bowie,
2007). This role for Sarm1 in producing cytokines was demonstrated through the reduced
RNA levels of several cytokines in a Sarm1-/- mouse in comparison to wild type levels (Lin
et al., 2013). This final clearance step, brought about by Schwann cells in the PNS and
macrophages systemically, is noteworthy as it signals the completion the degeneration
pathway, and clears the way for regeneration of the proximal nerve.
Conclusion
While recent discoveries, such as that of Highwire and Sarm1, have added to the
understanding of axonal degeneration, there are still many issues within the area to be dealt
with. Primarily, the development of a standard system for the comparison of protective
effects of different elements would be quite useful, due to the fact that, as discussed above,
overexpression or application of non-physiological levels of a protein/compound often serve
to confuse more than clarify whether and where that particular element is involved in
degeneration. Additionally, the elucidation of the missing steps within the above pathway, or
the structuring of a completely revised pathway following new evidence, will prove vital, as
it will allow directed therapeutic therapies to a range of neurodegenerative diseases to be
constructed. A final point to consider is whether, as previously discussed, Wallerian
degeneration is currently being unknowingly employed as an umbrella phrase for a range of
distinct pathways which share some key steps. It seems likely that this may be the case, but
experimentally proving this will allow future research to be more productive, as current
contradicting evidence, such as the protective effect of Wallenda in some neuron models,
may well be due to this fact.

24
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