Normal ControlMS LesionNAWM

Figure 3 Double immunofluorescent labeling using antibodies for calpain (green) and cell-specific marker (red) in normal control white matter, normal appearing white matter, and MS lesion. (A-C) Antibodies specific for cells expression MHC class II (L243). (D-F) Mononuclear phagocytes (EBM-11). Co-localization appears yellow (x 400). (Reproduced with permission from Shields et al., Proc. Natl. Acad. Sci. U. S. A. 96,11486-11491, 1999.)

Figure 3 Double immunofluorescent labeling using antibodies for calpain (green) and cell-specific marker (red) in normal control white matter, normal appearing white matter, and MS lesion. (A-C) Antibodies specific for cells expression MHC class II (L243). (D-F) Mononuclear phagocytes (EBM-11). Co-localization appears yellow (x 400). (Reproduced with permission from Shields et al., Proc. Natl. Acad. Sci. U. S. A. 96,11486-11491, 1999.)

spreading) (Shields and Banik, 1998a). In support of this hypothesis, we have recently demonstrated increased calpain expression (transcriptional, translational) in activated human lymphoid cell lines, which also secreted the enzyme (Deshpande et al., 1995a, 1995b). Furthermore, it is relevant to suggest that activated calpain secreted from MBP-spe-cific T-cells may be detrimental to neuronal, oligoden-droglial, and axonal preservation.

The degradation of 3P, NFP, and spectrin integrity, all of which maintain the architecture of axon and cell membrane, will lead to axonal degeneration and cell death in demyeli-nating diseases, as previously suspected (Newcombe et al., 1982), and axonal damage can be assessed by measuring increases in dephosphorylation of high molecular weight NFPs, which precede actual loss of NFP protein (Trapp et al., 1998), or by measuring changes in amyloid precursor protein transport (Kornek et al., 2000). Although our previous studies indicated that calpain activation, infiltration of cells, and upregulation of calpain expression in EAE spinal cord occurred after the appearance of clinical symptoms, they did not precisely determine at which point and in which cells calpain is detected first. Subsequent studies (Schaecher et al., 2002) demonstrated that calpain expression was increased in microglia/macrophages in EAE spinal cord at day 9 after induction of EAE (first day after disease onset); however, although there was a substantial increase in the number of activated T-cells. T-cells did not express calpain until day 10 (Fig. 4). In addition, degradation of MBP and NFP was increased, indicating that axonal damage was occurring after disease onset and suggesting that calpain production from infiltrating immune cells may contribute to axonal damage and neurodegeneration.

To better understand the timing of calpain-induced damage in EAE, we also examined calpain expression in neurons in spinal cord gray matter via immunohistochemical staining of control and EAE spinal cord using an antibody specific for m-calpain and a neuron-specific anti-NeuN antibody (Fig. 5). Calpain expression was negligible in neurons from control spinal cord tissue; however, a large number of neurons, as well as other cells, stained positive for calpain after onset of disease, indicating that EAE neurons do express calpain at an elevated level after onset of disease. Current preliminary studies conducted in our laboratory indicate that axonal damage and neuronal death correlate with increased calpain expression and activity in EAE spinal cord after onset of disease (Figs. 5 and 6).

Because optic neuritis (which results in impairment of vision) can be an early clinical manifestation of MS and because the majority of patients with optic neuritis develop

Figure 4 Double immunofluorescent labeling using antibodies for calpain (green) and cell-specific markers (red) over time. Control and EAE spinal cord were stained with T-cell (CD4) or macrophage/microglia (OX-42) antibodies at days 8—11. Co-localization appears yellow (x 400). (Reproduced with permission from Schaecher et al., J. Neuroimmunol. 129:1-9, 2002.)

Figure 4 Double immunofluorescent labeling using antibodies for calpain (green) and cell-specific markers (red) over time. Control and EAE spinal cord were stained with T-cell (CD4) or macrophage/microglia (OX-42) antibodies at days 8—11. Co-localization appears yellow (x 400). (Reproduced with permission from Schaecher et al., J. Neuroimmunol. 129:1-9, 2002.)

MS (Compston et al., 1991), it is likely that glial cells (oligodendrocytes) and axons in the optic nerve are damaged early in EAE, which is also used as an animal model for optic neuritis (Raine et al., 1980). As previously mentioned, calpain expression and activity are upregulated in EAE after onset of disease. Damage to retinal ganglion cells and visual impairment, as assayed by electroretinograms and

Figure 5 Double immunofluorescent staining using antibodies for calpain (green) and neuron specific marker (NeuN) in control EAE spinal cord. Co-localization appears yellow (x 400).

visual-evoked potentials, have been demonstrated in EAE (Meyer et al., 2001). This suggested that increased calpain might damage cells and axons in the optic nerve, leading to visual dysfunction. We have found that axons are damaged, as assessed by measuring increases in dephosphorylated NFP, before onset of clinical symptoms (Fig. 6). Increased calpain expression and apoptotic/necrotic glial cells also occurred before disease onset (Guyton et al., 2004a), suggesting that the optic nerve is affected early with loss of

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