Anna Jurewicz

Knockout of caspase-8, a cysteine protease that participates in the signaling for cell death by receptors of the TNF/nerve growth factor family, is lethal to mice in utero. To explore tissue-specific roles of this enzyme, we established its conditional knockout using the Cre/loxP recombination system. Consistent with its role in cell death induction, deletion of caspase-8 in hepatocytes protected them from Fas-induced caspase activation and death. However, application of the conditional knockout approach to investigate the cause of death of caspase-8 knockout embryos revealed that this enzyme also serves cellular functions that are nonapoptotic. Its deletion in endothelial cells resulted in degeneration of the yolk sac vasculature and embryonal death due to circulatory failure. Caspase-8 deletion in bone-marrow cells resulted in arrest of hemopoietic progenitor functioning, and in cells of the myelomonocytic lineage, its deletion led to arrest of differentiation into macrophages and to cell death. Thus, besides participating in cell death induction by receptors of the TNF/nerve growth factor family, caspase-8, apparently independently of these receptors, also mediates nonapoptotic and perhaps even antiapoptotic activities.

Inositol has 8 stereoisomers, four of which are physiologically active. <i>myo</i>-Inositol is the most abundant isomer in the brain and more recently shown that <i>epi</i>- and<i>scyllo</i>-inositol are also present. <i>myo</i>-Inositol complexes with Aβ42 <i>in vitro</i> to form a small stable micelle. The ability of inositol stereoisomers to interact with and stabilize small Aβ complexes was addressed. Circular dichroism spectroscopy demonstrated that <i>epi</i>- and <i>scyllo</i>- but not <i>chiro</i>-inositol were able to induce a structural transition from random to β-structure in Aβ42. Alternatively, none of the stereoisomers were able to induce a structural transition in Aβ40. Electron microscopy demonstrated that inositol stabilizes small aggregates of Aβ42. We demonstrate that inositol-Aβ interactions result in a complex that is non-toxic to nerve growth factor-differentiated PC-12 cells and primary human neuronal cultures. The attenuation of toxicity is the result of Aβ-inositol interaction, as inositol uptake inhibitors had no effect on neuronal survival. The use of inositol stereoisomers allowed us to elucidate an important structure-activity relationship between Aβ and inositol. Inositol stereoisomers are naturally occurring molecules that readily cross the blood-brain barrier and may represent a viable treatment for AD through the complexation of Aβ and attenuation of Aβ neurotoxic effects.

Multiple sclerosis (MS) is considered to be an autoimmune disease that is directed either at myelin or at its cell of origin, the oligodendrocytes (OL). The inflammatory lesions in the central nervous system contain multiple myelin Ag-restricted and nonrestricted cell populations with the potential to mediate tissue injury. Previous studies indicate that it is possible to generate MHC class I-restricted myelin peptide-specific cytotoxic CD8 T cells, and that human adult OLs express MHC class I molecules in vitro. The purpose of this study was to demonstrate that myelin basic protein peptide-specific CD8 T cells could induce OL injury. We generated CD8 T cell lines from six healthy donors and five MS patients, and all cell lines were HLA-A2 positive. The obtained CD8 cell lines induced lysis of HLA-A2- but not HLA-A3-transfected HMy2.C1R cells in the presence of myelin basic protein peptide 110-118. In the absence of exogenous peptide, the CD8 T cell lines were cytotoxic to HLA-A2 but not to non-HLA-A2 OLs. Cytotoxicity was blocked with anti-MHC class I-blocking Ab. These results support the postulate that autoreactive CD8 cytotoxic T cells can contribute to the tissue injury in MS.

Tumour necrosis factor (TNF)-induced death of oligodendrocytes, the cell type targeted in multiple sclerosis, is mediated by TNF receptor p55 (TNFR-p55). The ligation of TNFR-p55 induces several signal transduction pathways; however, the precise mechanism involved in human oligodendrocyte (hOL) death is unknown. We defined that TNF-induced death of hOLs is non-caspase dependent, as evidenced by lack of generation of caspases 8, 1 and 3 active subunits; lack of cleavage of caspases 1 and 3 fluorogenic substrates; and lack of hOL death inhibition by the general caspase inhibitor, ZVAD.FMK. Electrophoresis of TNF-exposed hOL DNA revealed large-scale DNA fragmentation characteristic of apoptosis-inducing factor (AIF)-mediated cell death, and co-localization experiments showed that AIF translocation to the nucleus occurred upon exposure to TNF. AIF depletion by an antisense strategy prevented TNF-induced hOL death. These results indicate that TNF-induced death of hOLs is dependent on AIF, information of significance for the design strategies to protect hOLs during immune-mediated demyelination.