Fas FasL

Fas is a member of the tumor necrosis factor (TNF) receptor superfamily that is expressed on target cells as a receptor for the Fas ligand (FasL) present on the surface of cytotoxic T-cells. The intracellular domain of Fas contains a death domain (DD) region necessary for induction of apop-tosis. Upon binding of FasL to Fas on a target cell, the DD is induced to recruit an adaptor protein called Fas-associated death domain (FADD). FADD bears both a death domain, via which it binds to Fas, and a death effector domain,

Figure 13 Chronically infected SJL/J mice exhibit profound loss of axons in the spinal cord. (A) Bielschowski-stained axons in uninfected, aged SJL/J mouse spinal cord. (B) Bielschowski-stained axons in the spinal cord of a chronically infected (180 days) SJL/J mouse. Note the loss of stained axons and the relative disarray of the axons that are present. (C) Brainstem neurons in an uninfected SJL/J mouse labeled in a retrograde fashion. Fluorogold was applied to the surface of a hemisectioned spinal cord. After recovery and adequate time for retrograde axonal transport of the fluorogold, brainstem and spinal cord were sectioned and analyzed for the presence of fluorogold within neurons of the brainstem. Uninfected animals exhibit reproducible and robust retrograde transport of the dye. (D) However, chronically infected SJL/J mice exhibit a profound defect in retrograde transport of fluorogold, suggesting that the descending axons of brainstem neurons are completely lost or dysfunctional. (E) Electron micrograph of a normal rubrospinal neuron in an uninfected SJL/J mouse. (F) Example of a degenerating rubrospinal neuron in a chronically infected SJL/J mouse. No such degenerating profiles were observed in the uninfected mouse. This finding suggests that the loss of retrograde transport and normal axon staining is correlated with loss of brainstem neurons. This may be the cellular substrate of the neurological deficits observed in these animals.

Figure 13 Chronically infected SJL/J mice exhibit profound loss of axons in the spinal cord. (A) Bielschowski-stained axons in uninfected, aged SJL/J mouse spinal cord. (B) Bielschowski-stained axons in the spinal cord of a chronically infected (180 days) SJL/J mouse. Note the loss of stained axons and the relative disarray of the axons that are present. (C) Brainstem neurons in an uninfected SJL/J mouse labeled in a retrograde fashion. Fluorogold was applied to the surface of a hemisectioned spinal cord. After recovery and adequate time for retrograde axonal transport of the fluorogold, brainstem and spinal cord were sectioned and analyzed for the presence of fluorogold within neurons of the brainstem. Uninfected animals exhibit reproducible and robust retrograde transport of the dye. (D) However, chronically infected SJL/J mice exhibit a profound defect in retrograde transport of fluorogold, suggesting that the descending axons of brainstem neurons are completely lost or dysfunctional. (E) Electron micrograph of a normal rubrospinal neuron in an uninfected SJL/J mouse. (F) Example of a degenerating rubrospinal neuron in a chronically infected SJL/J mouse. No such degenerating profiles were observed in the uninfected mouse. This finding suggests that the loss of retrograde transport and normal axon staining is correlated with loss of brainstem neurons. This may be the cellular substrate of the neurological deficits observed in these animals.

Figure 14 Brain-infiltrating lymphocytes were isolated from mice that were infected for 7 days with Theiler's virus. These cells were stained with fluorescently labeled tetramers that presented either the Theiler's virus immunodominant VP2121-130 peptide within the context of H-2Db or the irrelevant E7 peptide in the context of H-2Db. Using flow cytometry for analysis of cells positive for tetramer and CD8, we observed that 68% of CD8+ brain infiltrating lymphocytes were specific for the VP2121-130 peptide, but only 4% of such cells labeled with the irrelevant tetramer.

Figure 14 Brain-infiltrating lymphocytes were isolated from mice that were infected for 7 days with Theiler's virus. These cells were stained with fluorescently labeled tetramers that presented either the Theiler's virus immunodominant VP2121-130 peptide within the context of H-2Db or the irrelevant E7 peptide in the context of H-2Db. Using flow cytometry for analysis of cells positive for tetramer and CD8, we observed that 68% of CD8+ brain infiltrating lymphocytes were specific for the VP2121-130 peptide, but only 4% of such cells labeled with the irrelevant tetramer.

Figure 15 (A) Normally myelinated axons in the white matter of an infected C57BL/6 H-2b mouse. (B) Theiler's virus infection of per-forin-deficient C57BL/6 H-2b mice results in extensive demyelination. Despite such widespread demyelination, however, these animals do not exhibit significant functional impairment. This finding suggests that perforin-mediated killing by CD8+ T-cells is required for axon loss and functional deficits, but not for demyelination.

Figure 15 (A) Normally myelinated axons in the white matter of an infected C57BL/6 H-2b mouse. (B) Theiler's virus infection of per-forin-deficient C57BL/6 H-2b mice results in extensive demyelination. Despite such widespread demyelination, however, these animals do not exhibit significant functional impairment. This finding suggests that perforin-mediated killing by CD8+ T-cells is required for axon loss and functional deficits, but not for demyelination.

through which it associates with pro-caspase-8. Thus, a death-inducing complex, called the death-inducing signaling complex (DISC), is formed at the Fas receptor. Pro-cas-pase-8 recruitment to the DISC results in autocleavage and activation of caspase-8, which then goes on to activate effector caspases such as caspase-3, resulting in apoptosis (Thorburn, 2004).

The Fas/FasL pathway is active in essentially all cyto-toxic cells, but is apparently most important for CD4+ cells of the Th1 phenotype (Ju et al., 1994). Although cultured CD4+ and CD8+ T-cells can engage both the perforin and the Fas/FasL pathways, in vivo experiments suggest that MHC class I-dependent killing is dominated by the perforin pathway, whereas MHC class II-dependent elimination is almost entirely mediated by Fas/FasL interactions (Graubert et al., 1997; Schulz et al., 1995). Thus, a comparison between the relative role of perforin and Fas/FasL in demyelination and neurological deficit associated with Theiler's virus infection is a reasonable approach to address the CD8+ hypothesis.

Indeed, in support of the CD8+ hypothesis, and in contrast to the findings for perforin, the Fas/FasL system appears to play a minimal role in determining resistance to Theiler's virus infection. Neither the Ipr mutant, deficient in Fas, nor the gld mutant, defective in FasL binding, developed demyelination or neurological deficit in response to Theiler's virus infection (Murray et al., 1998). Moreover, Rensing-Ehl and colleagues have reported that neurons express only low levels of Fas, even after cytokine stimulation, and are resistant to FasL-mediated killing (Rensing-Ehl et al., 1996). In contrast, other investigators have suggested that in certain situations, MHC class I-restricted killing of neurons by virus-specific CD8+ T-lymphocytes is mediated through the Fas/FasL pathway, but not the perforin pathway (Medana et al., 2000). Likewise, the role of Fas/FasL in human MS is less clear than in the Theiler's virus model. Although one report indicates that Fas is irrelevant to oligo-dendrocyte loss in MS lesions (Bonetti and Raine, 1997), another provides evidence, at least in vitro, that Fas-mediated signaling contributes to a nonapoptotic injury of oligo-dendrocytes. Of particular interest, FasL has been shown to protect neurons against perforin-mediated cytotoxicity (Medana et al., 2001), raising the possibility that both these mechanisms of neuronal killing may interact with one another in as yet uncharacterized, and potentially extremely complicated, ways.

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