Eae In Mutant And Transgenic Animals

Mutant or transgenic animals provide a powerful tool for studying the physiological function of genes, whether they are overexpressed or absent. These methods enable us to target the genes studied into the relevant organs. One example of such manipulation is the transgenic mice that overexpress in the anti-apoptotic gene bcl-xl in their T-cells. These mice demonstrate an earlier onset and a chronic form of EAE.22 Another study with mice deficient in Fas, the pro-apoptotic gene, shows a more severe clinical course of the disease.23 In our study, we investigated the possible role of axonal damage and the role of anti-apoptotic regulation of neurons in the patho-genesis of MS. We used transgenic mice that overexpress the human bcl-2 gene exclusively in their neurons, under the control of neuron-specific enolase (NSE) promoter. In previous studies, Bernard et al24 have demonstrated that these mice contain increased numbers of neurons, due to greater resistance to neuronal death during development. Other studies have shown that these mice have a better performance in several behavioral assays.25 The NSE-bcl-2 mice are resistant to induced ischemia and to cell death induced by glutamate and free radicals.26 The most important characteristic in the present context is their axonal resistance to crash and axotomy.27 To study the effects of Bcl-2 overexpression in neurons on the pathogenesis of EAE, we compared the clinical manifestations of the disease in WT (C57BL/6) and transgenic NSE-bcl-2 mice,28 following disease induction with pMOG35-55. We found that, 2 weeks after encephalitogenic challenge, WT mice developed severe EAE, characterized by complete hind limb paralysis (mean maximal score of 2.61 ± 0.18). In contrast, the NSE-bcl-2 mice were significantly resistant to MOG-induced EAE. Half of the immunized transgenic mice remained disease-free, while the other half demonstrated mild clinical signs characterized by loss of tail tonicity and some weakness of the hind limbs, compared to WT mice (mean maximal score of 1.56 ± 0.39, P = 0.017). Thus, the disease induced in NSE-bcl-2 mice was markedly reduced both in incidence and clinical severity.

Further characterization of spinal cords and brains of the WT animals revealed a widespread perivascular lymphohistiocytic inflammatory infiltrate with scattered neutrophils accompanied by severe demyelination and axonal damage. In contrast, sections of healthy NSE-bcl-2 mice showed only focal perivascular lymphohistiocytic inflammation. Bielshowsky staining of spinal cord sections from immunized WT mice showed severe axonal damage. Only minimal axonal damage in regions surrounding inflammation was found in healthy immunized NSE-bcl-2 mice.

To rule out the possibility that the differences in clinical manifestations of the disease in the NSE-bcl-2 mice were due to generalized immune dysfunction associated with Bcl-2 overexpression in their neurons, the ability of the NSE-bcl-2 mice to mount a T-cell response was compared to that of WT mice. NSE-bcl-2 and WT mice were immunized with pMOG35-55 and their recall T-cell proliferative response was compared. The immune potency of T-cells in developing a delayed-type hypersensitivity (DTH) response was assayed in both groups. We found that the DTH response against pMOG35-55 or against pertussis protein derivative (PPD) was similar in both WT and NSE-bcl-2 mice. These two experiments indicate that there are no differences between these groups in their capacity to elicit functional encephali-togenic T-cells specific for pMOG35-55. We then compared free radical production in brain synapto-somes of WT and NSE-bcl-2 mice following an oxida-tive burst by dechlorofluorocin (DCFH), and found that overexpression of Bcl-2 is associated with an increased free radical scavenger capacity of the synap-tosomes.

In both EAE and MS, the inflammatory process in the CNS plays an important role in demyelination and axonal damage, consequently contributing to the disease progression. Such inflammatory changes are associated with the local production of highly destructive agents, including nitric oxide (NO) and reactive oxygen species (ROS), that may contribute to axonal damage.29,30 It has been shown that in rodents with EAE, non-specific inhibitors of NO synthetase (NOS) partially ameliorate the disease.31 It was suggested that the mechanism by which NO kills cells is not yet understood. However, it appears that NO, together with superoxide, forms highly toxic peroxynitrate, which, in turn, may generate additional free radicals with harmful effects, including lipid and protein oxidation.32 Our study demonstrated that purified synaptosomes from CNS tissues of NSE-bcl-2 mice produced significantly fewer free radicals than those of WT mice when challenged with H2O2 and NO.28 Indeed, it has already been shown that oxidative stress is important in the pathogenesis of MBP-induced EAE, and treatment with free radical scavengers such as N-acetylcysteine inhibits disease development.33

Although the exact mechanism by which Bcl-2 protects cells from apoptotic death is not yet fully understood, it has been suggested that its anti-

apoptotic effects are exerted by promoting the function of antioxidants via unknown mechanisms.34-35 Several in vitro and in vivo studies have demonstrated that Bcl-2 protects cells from apopto-sis induced by endogenous or exogenous oxidants and reduces ROS-induced damage.36-37

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