Molecular Axon Components

Although a number of molecules, or classes of molecules, secreted by activated immune cells have been implicated in axon injury in MS, the early events leading to transection of the axon have not been studied in any depth. The appearance of the axon end-bulb already indicates that axon injury has proceeded to an irreversible state. The sealing of the ends of an injured axon involves calcium-dependent processes including activation of calpain and phospholipase A2 (Howard et al., 1999; Geddis and Rehder, 2003), but we know little of the molecular events that precede this catastrophic event.

1. Active Axon Degeneration

Axon degeneration accompanies an extraordinary diversity of injury to, and diseases of, the nervous system, and a favored model to study the mechanisms that underlie axon degeneration has been the study of degeneration of the distal segment of a transected axon, wallerian degeneration. The morphological events that appear in wallerian degeneration are found at the end stage of almost every form of axon degeneration, suggesting that there is a final common pathway in the degeneration process (Griffin et al., 1996). It was long thought that the axon degenerated as a consequence of separation from the cell body leading to a withering of the distal process, an essentially passive process (Finn et al., 2000). The degeneration of the axon was also linked in acute transection to an influx of calcium that activated calcium-

activated proteases within the axoplasm, which in turn degraded the axon cytoskeletal elements (Schlaepfer and Hasler, 1979).

The serendipitous discovery of a strain of mouse carrying a mutation that dramatically slows wallerian degeneration (Lunn et al., 1989) has radically changed our views of how axons degenerate (Finn et al., 2000; Coleman and Perry, 2002). In wild-type mice, and indeed all mammals, the axon segment distal to an injury normally undergoes wallerian degeneration within a few days of transection. In the mutant Wlds mice the axons of both CNS and PNS neurons may survive for several weeks separated from the cell body (Fig. 3). This simple observation tells us that axon degeneration in wild-type axons must be an active autodestructive process akin to programmed-cell-death or apopto-sis of the cell body of neurons and other cells. The mutation in these mice has now been identified, and the Wlds gene was found to be a chimeric protein formed from UbE4b, an ubiquitin ligase, and nicotinamide mononucleotide ade-nylyltransferase (Nmnat), an NAD synthesizing enzyme (Mack et al., 2001). The mechanism of action of this gene is not known, but it clearly implicates the ubiquitin-protea-some pathway in axon degeneration (see later).

The programmed-cell-death-like process in axons shares some similarities with the apoptotic cascades described at the neuronal cell soma, but there are also notable differences. Transected neurites undergoing wallerian degeneration in vitro express phosphatidylserine residues on the plasma membrane as detected by Annexin V undergo bleb-bing, and lose their mitochondrial potential before degeneration (Sievers et al., 2003). Manipulations that protect the cell bodies of some neurons from apoptosis, such as raising extracellular potassium or intracellular cyclic adenosine monophosphate, also protect transected neurites in vitro (Buckmaster et al, 1995). However, the degeneration of neu-rites in vitro is not protected by caspase inhibitors (Finn et al., 2000; Sievers et al, 2003), and axons in vivo are not protected by overexpression of Bcl-2 (Burne et al., 1996), although they are protected in Bax deficient mice (Dong et al 2003; J.W. McDonald, personal communication).

Thus, the "molecules of destruction" secreted by inflammatory cells in the MS lesion act on axons that contain the biochemical machinery that maintains the balance between life and death of the axon. These pathways act in an analogous fashion to, but distinct from, the pro-apoptic and anti-apoptotic pathways maintaining the balance between life and death of the cell soma. It is important to note that the protection of the axon in wallerian degeneration by the Wlds gene has now been shown to extend to protection of axons subjected to taxol toxicity (Wang et al. 2003), to protection of axons in a mouse model of motorneuron disease (Ferri et al., 2003), and protection of axons from the axon degeneration present in a dysmyelinating mutant, the Po knockout mouse (Samsam et al., 2003). A simple hypothesis is that secretory

Wallerian Degeneration Axons

Figure 3 The remarkable phenotype of the Wlds mouse (Lunn et al., 1989). Central panel (B) illustrates the appearance of the normal sciatic nerve in a 1-|m semithin section stained with toluidine blue. Panel (C) illustrates the appearance of the distal nerve of a wild-type mouse nerve undergoing wallerian degeneration 7 days after transection. Panel (A) shows the remarkable preservation of the distal axon segment 7 days after sciatic nerve transection in the Wlds mouse.

Figure 3 The remarkable phenotype of the Wlds mouse (Lunn et al., 1989). Central panel (B) illustrates the appearance of the normal sciatic nerve in a 1-|m semithin section stained with toluidine blue. Panel (C) illustrates the appearance of the distal nerve of a wild-type mouse nerve undergoing wallerian degeneration 7 days after transection. Panel (A) shows the remarkable preservation of the distal axon segment 7 days after sciatic nerve transection in the Wlds mouse.

products of inflammatory cells, or contact with inflammatory cells, leads to local activation of these autodestruction pathways in the axon, and thus, a local axon transection.

2. Axon Calcium Levels

The loss of Ca2+ homeostasis in the axon has long been proposed as an early event in axon degeneration (Schlaepfer and Hasler, 1979). Alterations in ion homeostasis leading to increased permeability of the axolemma allows intra-axonal Ca2+ to rise, and reversal of the Na+-Ca2+ transporter has also been implicated in axon injury in ischemia (Stys, 1998) (Chapter 19). A rise in intra-axonal Ca2+ can activate proteases, in particular calpains (see Chapter 20), which in turn play a role in the degradation of the axon cytoskeleton. Although it is possible that changes in intra-axonal Ca2+ levels are critical in axon injury in MS lesions, there is no direct information on the link between inflammation and Ca2+ homeostasis in axons.

3. Caspases

The caspases are key players in the apoptotic execution of many different types of cells including neurons (Friedlander, 2003); however, the role of the caspases in axon degeneration is unclear. Caspase activation and the release of cytochrome-c have been described in axons after traumatic injury (Buki et al., 2000) and in the spinal cord of animals with EAE (Ahmed et al., 2002). It has been suggested that the caspases could play a role in degradation of the cytoskeleton. On the other hand, it has been shown that caspase inhibitors do not prevent axon or neurite degeneration in studies of wallerian degeneration in vitro (Finn et al., 2000; Sievers et al., 2003). However, there are a number of routes to programmed cell death that are caspase-independ-ent (Jaattela and Tschopp, 2003), and these pathways have yet to be explored in axon degeneration.

4. Ubiquitin-Proteasome Pathway

The role of the ubiquitin proteasome pathway (UPP) in axon degeneration has been brought to the fore by the discovery of two spontaneously occurring mutant strains of mice. The phenotype of the gracile axon dystrophy mouse (gad) is spontaneous axon degeneration in the gracile nucleus of the midbrain (Mukoyama et al., 1989), which leads to a progressive degeneration of the sensory neurons. The mutation was found to be in a gene encoding the ubiq-uitin hydrolase, UCH-L1, an enzyme involved in the release of ubiquitin from its substrate (Saigoh et al., 1999). This enzyme is one of the most abundant cytoplasmic enzymes in neurons, but it is not known how loss of this enzyme activity results in axon degeneration. In contrast, the Wlds mouse was found to have a phenotype in which axons and synapses of both the CNS and PNS exhibited very slow wallerian degeneration (see Coleman and Perry, 2002, for review). The Wlds gene was found to be a chimeric gene affecting the function of the ubiquitin ligase UbE4b (Mack et al., 2001). This ligase is normally involved in the selection of substrates for ubiquitination before processing in the protea-some. The vertebrate-specific 300 amino acid N-terminal domain is essential for ubiquitin chain extension of the substrate by the ligase (Mahoney et al., 2002). The active site of the ligase U-box region has been replaced in the chimeric protein by the enzyme Nmnat. It seems possible that the fusion between the first 70 amino acids of UbE4b and the entire open-reading frame of Nmnat acts as a dominant negative inhibitor of normal UbE4b activity. Transgenic mice expressing the chimeric Wlds gene show a dose-dependent delay in wallerian degeneration (Mack et al., 2001), whereas transgenic mice expressing only the Nmnat portion of the chimeric gene do not show this phenotype (Coleman and Perry, 2002). The role of UbE4b in the axon is not known, and indeed in Wlds mice the Wlds protein is only detectable in the neuronal nucleus and not in the axon (Feng, Coleman, and Perry, unpublished observations).

Further evidence to support the role of the UPP in axon degeneration comes from in vitro studies using proteasome inhibitors (Zhai et al., 2003). These authors showed that axon and neurite degeneration could be delayed by application of proteasome inhibitors in vivo and in vitro. It should be noted, however, that the delay in degeneration was hours rather than the delay of several days induced by the Wlds gene in vitro (Buckmaster et al., 1995) or weeks in vivo (Perry et al., 1991). The role of the proteasome in neuronal survival is complex, as inhibitors of the proteasome may also induce neuronal apoptosis (Qiu et al., 2000).

5. Axonal Transport

The axon and its terminals are maintained in a state of homeostasis by transport systems that move materials in an anterograde and retrograde direction. The family of molecules responsible for anterograde movement of organelles and proteins are the kinesins, and some 38 members of this family have been detected in the brain (Miki et al., 2001). How inflammatory molecules might affect these transport systems has not been studied, and this potentially interesting and important area of research has been largely neglected. In a guinea pig model of EAE, despite a loss of only 25% of the axons, there is a 75% decrement in the amount of material transported along the optic nerve (Guy et al., 1989). Evidence also suggests that raised levels of glutamate may interfere with fast axonal transport (Ackerley et al., 2000), and treatment of mice with riluzole in MOG-induced EAE was found to reduce axon damage (Gilgun-Sherki et al., 2003). If axon transport processes can be affected by inflammatory mediators or glutamate, the consequences may not be immediate. In patients with Charcot-Marie-Tooth type 2a, patients develop a progressive late-onset peripheral neuropathy. The mutation underlying this disease is a mutation in one of the genes of the kinesin family, KIF-1B, a transporter involved in the movement of mitochondria (Zhao et al., 2001).

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