Immune Mediated Mechanisms of Axonal Loss

The exact molecular mechanisms of axonal loss in MS are unknown. The development of future neuroprotective therapies is not only critically dependent on our elucidating the cellular and molecular mechanisms of axonal loss in MS, but also on comprehending the interplay of these mechanisms with disease progression. Complicating our ability to interpret the pathophysiology of axonal injury is the probability that there are multiple mechanisms of axonal degeneration, which are dependent on the stage of the disease (Kornek and Lassmann, 1999; Bjartmar and Trapp, 2003). The link between disease activity and CNS inflammation is highly correlated, even when the disease is subclinical (Rudick et al., 1999b). Further, evidence supporting a link between inflammation and axonal pathology in MS patients was indicated by the positive correlation between inflammatory activity of cerebral MS lesions and axonal damage (Ferguson et al., 1997; Trapp et al., 1998; Kornek et al., 2000, 2001; Bitsch et al., 2000; Geurts et al., 2003). Additionally, axonal damage identified by increased levels of the light subunit of neurofilament proteins was detected in 78% of the CSF samples from RR-MS patients and demonstrated to correlate with clinical disability (Lycke et al., 1998). MRI studies provide further support for the dependence of axonal pathology on inflammation. For example, gadolinium-enhancing lesions on MRI scans have linked progressive brain atrophy with inflammation in RR-MS patients (Zivadinov and Zorzon, 2002). Previous reports have correlated CNS atrophy, as measured by MRI, with clinical disability suggesting that atrophy may represent axonal loss (Losseff and Miller, 1998). Of interest are the findings of progressive atrophy and persistent inflammation identified as gadolinium-enhancing lesions on MRI scans, which can be documented on subsequent MRI examinations, occurring even in the absence of clinical symptoms in patients with RR-MS (Simon et al., 1998, 1999; Rudick et al., 1997). These observations indicate that even during so-called stages of clinical remission, MS is progressing silently.

The strong correlation between inflammation and axonal transection suggests that axonal transection is immunemediated. Specific immunological attack on the axon has been considered as a possible mechanism of axonal degeneration in MS. Macrophages and activated microglia have been detected in close association with the end bulbs of transected axons in MS lesions using confocal microscopy (Fig. 4A, B) (Trapp et al., 1998). In addition, these cells sometimes contain neurofilament-positive inclusions (Fig. 4A). These observations suggest that macrophages and activated microglia may be targeting axons. Whether these cells are directly attacking axons or only removing axonal debris remains to be determined. Direct immunological targeting of axons is not without precedence. In acute motor axonal neuropathy (AMAN), a variant of Guillain-Barré syndrome (GBS), which is an autoimmune disease of the peripheral nervous system, primary immune-mediated attack on axons has been identified as the mechanism responsible for axonal degeneration (Ho et al., 1998). Antibody binding to an axolemmal protein has been demonstrated by the identification of immunoglobulins and the complement activation marker C3d at the nodes of Ranvier (Hafer-Macko et al., 1996). Macrophages then target the nodes of Ranvier, possibly in response to complement-derived chemoattractant signals such as C5a, and invade the periaxonal space, displacing myelin internodes and promoting axonal degeneration. Unlike AMAN, localization of antibodies specific to axonal components in the CNS has not been identified in MS lesions (Hafer-Macko et al., 1996; Ho et al., 1998).

Figure 4 Macrophages and microglia associate with transected axons (green) in active MS lesions. Macrophages (red, A) and microglia (red, B) surround and engulf terminal axonal swellings (large arrows), but have no consistent association with normal-appearing axons (arrowheads) or swellings in nontransected axons (small arrow). (Confocal micrographs reproduced from Trapp et al., 1998, with permission.)

Figure 4 Macrophages and microglia associate with transected axons (green) in active MS lesions. Macrophages (red, A) and microglia (red, B) surround and engulf terminal axonal swellings (large arrows), but have no consistent association with normal-appearing axons (arrowheads) or swellings in nontransected axons (small arrow). (Confocal micrographs reproduced from Trapp et al., 1998, with permission.)

In addition, an immunological attack directed at axons seems unlikely since most axons survive the acute demyeli-nating process. However, recent observations implicating cytotoxic CD8+ T-cells as mediators of axonal transection in inflammatory lesions were made in MS tissue (Babbe et al., 2000; Skulina et al., 2004), in EAE mice (Huseby et al., 2001), and in vitro (Medana et al., 2001; Giuliani et al., 2003). Other recent studies have reported that immunemediated mechanisms might target axonal subpopulations (Ganter et al., 1999; Lovas et al., 2000; Evangelou et al., 2001). In addition, antibodies to enriched fractions of axolemma have been detected in the CSF and serum of MS patients (Rawes et al., 1997). While evidence supporting direct immunological attack of axons in MS is scant, we should not overlook the possibility that it contributes to axon loss in MS.

Although evidence for cell-mediated mechanisms of axonal transection is inconclusive, loss of axons may be caused by nonspecific damage resulting from the inflammatory process. The inflammatory microenvironment contains a multitude of substances produced by activated immune and glial cells that potentially injure axons, including prote-olytic enzymes such as matrix metalloproteases, cytokines, oxidative products, and free radicals (Hohlfeld, 1997). In addition to the direct effects of inflammatory mediators, recent studies have indicated that inflammation induces aberrant glutamate homeostasis (Piani et al., 1991; Werner et al., 2001) and production of nitric oxide (NO) (Bo et al., 1994; Smith and Lassmann, 2002) in MS. Damage to mito-chondrial DNA and impaired activity of mitochondrial enzyme complexes of the electron transport chain mediated by oxidative radicals in MS lesions indicate that inflammation can affect energy metabolism, adenosine triphosphate (ATP) synthesis, and the viability of affected cells (Lu et al., 2000). Treatment with NBQX, an AMPA/kinate glutamate receptor antagonist, resulted in increased oligodendrocyte survival and reduced axonal damage in EAE, an animal model of MS (Pitt et al., 2000). The reduced axonal damage and increased oligodendrocyte survival were not due to reductions in inflammatory activity because NBQX has no anti-inflammatory effects; therefore NBQX's beneficial effects suggest that glutamate may also be involved in tissue damage in acute lesions.

Acute axonal damage may also occur via mechanical compression caused by increased extracellular pressure from inflammation-induced edema. Severe swelling in the CNS can lead to herniation or compression damage. Spinal cord axons may be more susceptible to compression damage, because they are anatomically located on the outside of the cord and have less space in the vertebrae to expand compared to the brain in the skull. In relapsing-remitting EAE mice, there was a 9% increase in spinal cord cross-sectional area during the first attack, which returned to normal at endstage disease (Wujek et al., 2002). Inflammation would also result in the disruption of normal vascular function, which could lead to ischemic-mediated axonal damage. An analogous mechanism for ischemic-induced axonal damage is proposed in GBS. Edema in GBS is thought to cause increased endoneurial fluid pressure and disruption of the nerve's vascular supply, resulting in ischemia-mediated axonal degeneration (Powell and Myers, 1996). Intriguing evidence for the activation of molecules involved in protecting against hypoxia/ischemia in MS brains has been reported (Graumann et al., 2003; Lassmann, 2003). The exact mechanism of inflammatory-mediated axonal loss is unknown; however, inflammation strongly correlates with axonal transection and is likely a major contributor responsible for accumulating axonal pathology during early stages of MS. Therefore, in addition to reducing inflammation and demyelination, aggressive anti-inflammatory treatment during RR-MS should also prevent axonal transection.

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