Abstract

Wallerian degeneration is a process that results when a nerve fiber is cut or crushed and the part of the axon distal to the injury (i.e. farther from the neuron's cell body) degenerates. This is also known as anterograde or orthograde degeneration. A related process known as 'Wallerian-like degeneration' occurs in many neurodegenerative diseases, especially those where axonal transport is impaired. Primary culture studies suggest that a failure to deliver sufficient quantities of the essential axonal protein NMNAT2 is a key initiating event.

Wallerian degeneration occurs after axonal injury in both the peripheral nervous system (PNS) and central nervous system (CNS). It occurs in the axon stump distal to a site of injury and usually begins within 24–36 hours of a lesion. Prior to degeneration, distal axon stumps tend to remain electrically excitable. After injury, the axonal skeleton disintegrates, and the axonal membrane breaks apart. The axonal degeneration is followed by degradation of the myelin sheath and infiltration by macrophages. The macrophages, accompanied by Schwann cells, serve to clear the debris from the degeneration.

Schwann cells respond to loss of axons by extrusion of their myelin sheaths, downregulation of myelin genes, dedifferentiation and proliferation. They finally align in tubes (Büngner bands) and express surface molecules that guide regenerating fibers. Within 4 days of the injury, the distal end of the portion of the nerve fiber proximal to the lesion sends out sprouts towards those tubes and these sprouts are attracted by growth factors produced by Schwann cells in the tubes. If a sprout reaches the tube, it grows into it and advances about 1 mm per day, eventually reaching and reinnervating the target tissue. If the sprouts cannot reach the tube, for instance because the gap is too wide or scar tissue has formed, surgery can help to guide the sprouts into the tubes. Regeneration is efficient in the PNS, with near complete recovery in case of lesions that occur close to the distal nerve terminal. However recovery is hardly observed at all in the spinal cord. One crucial difference is that in the CNS, including the spinal cord, myelin sheaths are produced by oligodendrocytes and not by Schwann cells.

History

Wallerian degeneration is named after Augustus Volney Waller. Waller experimented on frogs in 1850, by severing their glossopharyngeal and hypoglossal nerves. He then observed the distal nerves from the site of injury,

which were separated from their cell bodies in the brain stem.

Waller described the disintegration of myelin, which he referred to as "medulla", into separate particles of various sizes. The degenerated axons formed droplets that could be stained, thus allowing studies of the course of individual nerve fibres.

Axonal degeneration

Although most injury responses include a calcium influx signaling to promote resealing of severed parts, axonal injuries initially lead to acute axonal degeneration (AAD), which is rapid separation of the proximal (the part nearer the cell body) and distal ends within 30 minutes of injury. Degeneration follows with swelling of the axolemma, and eventually leads to bead like formation. The process takes roughly 24 hours in the PNS, and longer in the CNS. The signaling pathways leading to axolemma degeneration are currently unknown. However, research has shown that this AAD process is calcium–independent.

Granular disintegration of the axonal cytoskeleton and inner organelles occurs after axolemma degradation. Early changes include accumulation of mitochondria in the paranodal regions at the site of injury. Endoplasmic reticulum degrades and mitochondria swell up and eventually disintegrate. The depolymerization of microtubules occurs and is soon followed by degradation of the neurofilaments and other cytoskeleton components. The disintegration is dependent on Ubiquitin and Calpain proteases (caused by influx of calcium ion), suggesting that axonal degeneration is an active process and not a passive one as previously misunderstood. Thus the axon undergoes complete fragmentation. The rate of degradation is dependent on the type of injury and is also slower in the CNS than in the PNS. Another factor that affects degradation rate is the diameter of the axon: larger axons require a longer time for the cytoskeleton to degrade and thus take a longer time to degenerate.

Myelin clearance

Myelin is a phospholipid membrane that wraps around axons to provide them with insulation. It is produced by Schwann cells in the PNS, and by oligodendrocytes in the CNS. Myelin clearance is the next step in Wallerian degeneration following axonal degeneration. The cleaning up of myelin debris is different for PNS and CNS.

PNS is much faster and efficient at clearing myelin debris in comparison to CNS, and Schwann cells are the primary cause of this difference.

Another key aspect is the change in permeability of the blood-tissue barrier in the two systems.

In PNS, the permeability increases throughout the distal stump, but the barrier disruption in CNS is limited to

just the site of injury.

Myelin clearance | Clearance in PNS

The response of Schwann cells to axonal injury is rapid. The time period of response

is estimated to be prior to the onset of axonal degeneration. Neuregulins are believed

to be responsible for the rapid activation. They activate ErbB2 receptors in the Schwann

cell microvilli, which results in the activation of the mitogen-activated protein kinase

(MAPK).

Although MAPK activity is observed, the injury "sensing" mechanism of Schwann cells is

yet to be fully understood. The "sensing" is followed by decreased synthesis of myelin lipids

and eventually stops within 48 hrs. The myelin sheaths separate from the axons at

the Schmidt-Lanterman incisures first and then rapidly deteriorate and shorten to form

bead-like structures. Schwann cells continue to clear up the myelin debris by degrading their

own myelin, phagocytose extracellular myelin and attract macrophages to myelin debris for further phagocytosis. However, the macrophages are not attracted to the region for the first few days; hence the Schwann cells take the major role in myelin cleaning until then.

Schwann cells have been observed to recruit macrophages by release of cytokines and

chemokines after "sensing" of axonal injury. The recruitment of macrophages helps

improve the clearing rate of myelin debris. The resident macrophages present in the

nerves release further chemokines and cytokines to attract further macrophages.

The degenerating nerve also produce macrophage chemotactic molecules.

Another source of macrophage recruitment factors is serum. Delayed macrophage recruitment

was observed in B-cell deficient mice lacking serum antibodies. These signaling molecules together cause an influx of macrophages, which peaks during the third week

after injury. While Schwann cells mediate the initial stage of myelin debris clean up,

macrophages come in to finish the job. Macrophages are facilitated by opsonins, which label debris for removal.

The 3 major groups found in serum include complement, pentraxins, and antibodies. However, only complement has shown to

help in myelin debris phagocytosis.

Murinson et al. (2005) observed that non-myelinated or myelinated Schwann cells in contact with an injured

axon enter cell cycle thus leading to proliferation. Observed time duration for

Schwann cell divisions were approximately 3 days after injury.

Possible sources of proliferation signal are attributed to the ErbB2 receptors and the ErbB3 receptors.

This proliferation could further enhance the myelin cleaning rates and plays an essential role in

regeneration of axons observed in PNS. Schwann cells emit growth factors that attract new axonal sprouts growing from the proximal stump after complete degeneration of the injured distal stump.

This leads to possible reinnervation of the target cell or organ. However, the reinnervation is

not necessarily perfect, as possible misleading occurs during reinnervation of the proximal axons to target cells.

Myelin clearance | Clearance in CNS

In comparison to Schwann cells, oligodendrocytes require axon signals to survive.

In their developmental stages, oligodendrocytes that failed to make contact to axon and

receive any axon signals underwent apoptosis.

Experiments in Wallerian degeneration have shown that upon injury oligodendrocytes either undergo programmed cell death or enter a state of rest. Therefore, unlike Schwann cells, oligodendrocytes fail to clean up the myelin sheaths and their debris. In experiments conducted on rats

myelin sheaths were found for up to 22 months. Therefore, CNS rates of myelin sheath clearance are very slow and

could possibly be the cause for hindrance in the regeneration capabilities of the CNS axons as no growth factors

are available to attract the proximal axons. Another feature that results eventually is Glial scar formation.

This further hinders chances for regeneration and reinnervation.

Oligodendrocytes fail to recruit macrophages for debris removal.

Macrophage entry in general into CNS site of injury is very slow. In contrast to PNS,

Microglia play a vital role in CNS wallerian degeneration. However, their recruitment is

slower in comparison to macrophage recruitment in PNS by approximately 3 days. Further, microglia might be activated but hypertrophy, and fail to transform

into fully phagocytic cells. Those microglia that do transform, clear out the debris effectively. Differentiating phagocytic microglia

can be accomplished by testing for expression of Major histocompatibility complex (MHC) class I and II during wallerian degeneration.

The rate of clearance is very slow among microglia in comparison to macrophages. Possible source for variations in clearance rates could include lack of

opsonin activity around microglia, and the lack of increased permeability in the blood–brain barrier.

The decreased permeability could further hinder macrophage infiltration to the site of injury.

These findings have suggested that the delay in Wallerian degeneration in CNS in comparison to PNS is

caused not due to a delay in axonal degeneration, but rather is due to the difference in clearance rates of myelin in CNS and PNS.

Regeneration

Regeneration follows degeneration. Regeneration is rapid in PNS, allowing for rates of up to 1 millimeter a day of regrowth. Grafts may also be needed to allow for appropriate reinnervation.

It is supported by Schwann cells through growth factors release. CNS regeneration is much slower, and is almost absent in most vertebrate species.

The primary cause for this could be the delay in clearing up myelin debris. Myelin debris, present in CNS or PNS, contains several

inhibitory factors. The prolonged presence of myelin debris in CNS could possibly hinder the regeneration.

An experiment conducted on Newts, animals that have fast CNS axon regeneration capabilities, found that

Wallerian degeneration of an optic nerve injury took up to 10 to 14 days on average, further suggesting that slow clearance

inhibits regeneration.

Regeneration | Schwann cells and endoneural fibroblasts in PNS

In healthy nerves, Nerve growth factor (NGF) is produced in very small amounts. However, upon injury, NGF mRNA expression increases

by five to sevenfold within a period of 14 days. Nerve fibroblasts and Schwann cells play an important role in increased expression of

NGF mRNA.

Macrophages also stimulate Schwann cells and fibroblasts to produce NGF via macrophage-derived interleukin-1.

Other neurotrophic molecules produced by Schwann cells and fibroblasts together include Brain-derived neurotrophic factor, Glial cell line-derived neurotrophic factor,

Ciliary neurotrophic factor, Leukemia inhibitory factor, Insulin-like growth factor, and Fibroblast growth factor. These factors

together create a favorable environment for axonal growth and regeneration. Apart from growth factors, Schwann cells

also provide structural guidance to further enhance regeneration. During their proliferation phase, Schwann cells begin to form a line of cells called

"Bands of Bungner" within the basal laminar tube. Axons have been observed to regenerate

in close association to these cells.

Schwann cells upregulate the production of cell surface adhesion molecule ninjurin further

promoting growth.

These lines of cell guide the axon regeneration in proper direction.

The possible source of error that could result from this is possible mismatching of the

target cells as discussed earlier.

Due to lack of such favorable promoting factors in CNS, regeneration is stunted in CNS.

Delayed Wallerian degeneration

Mice belonging to the strain C57BL/"Wld" have delayed Wallerian degeneration,

and, thus, allow to study the roles of various cell types and the underlying cellular and molecular processes. Current understanding of the

process has been possible via experimentation on the "Wld" strain of mice. The mutation occurred first in mice in

Harlan-Olac, a laboratory producing animals the United Kingdom. The "Wld" mutation

is an autosomal-dominant mutation occurring in the mouse chromosome 4. The gene mutation is an 85-kb tandem triplication, occurring naturally.

The mutated region contains two associated genes: nicotinamide mononucleotide adenlyl transferase 1 (Nmnat-1) and ubiquitination factor e4b (Ube4b).

A linker region encoding 18 amino acids is also part of the mutation.

The protein created, localizes within the nucleus and is undetectable in axons.

Delayed Wallerian degeneration | Effects of mutation

The mutation causes no harm to the mouse. The only known effect is that the Wallerian degeneration

is delayed by up to three weeks on average after injury of a nerve. At first, it was suspected that the

"Wld" mutation slows down the macrophage infiltration, but recent studies suggest that the mutation protects axons rather than

slowing down the macrophages. The process by which the axonal protection is achieved is

poorly understood. However, studies suggest that the "Wld" mutation leads to overexpression of the Nmnat-1 protein, which

leads to increased NAD synthesis. This in turn activates

SIRT1-dependent process within the nucleus, causing changes in gene transcription.

NAD by itself provides added axonal protection by increasing the axon's energy resources. More recent work, however, raises doubt that either NMNAT or NAD can substitute for the full length "Wld" gene. These authors demonstrated by both in vitro and in vivo methods that the protective effect of overexpression of NMNAT1 or the addition of NAD did not protect axons from degeneration. Thus, the underlying biological mechanism accounting for the "Wld" phenotype remains unknown.

The provided axonal protection delays the onset of Wallerian degeneration. Schwann cell activation

would be delayed, and they would not detect axonal degradation signals from ErbB2 receptors. In experiments on "Wld" mutated mice,

macrophage infiltration was considerably delayed by up to six to eight days.

However, once the axonal degradation has begun,

degeneration takes its normal course, and, respective of the nervous system, degradation follows at the above-described rates.

Possible effects that could result due to this late onset would be weaker regenerative abilities in the mice.