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. myo-Inositol is the most abundant isomer in the brain and more recently shown that epi-and scyllo-inositol are also present. myo-Inositol complexes with A␤42 in vitro to form a small stable micelle. The ability of inositol stereoisomers to interact with and stabilize small A␤ complexes was addressed. Circular dichroism spectroscopy demonstrated that epi-and scyllo-but not chiro-inositol were able to induce a structural transition from random to ␤-structure in A␤42. Alternatively, none of the stereoisomers were able to induce a structural transition in A␤40. Electron microscopy demonstrated that inositol stabilizes small aggregates of A␤42. We demonstrate that inositol-A␤ 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 A␤-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 A␤ 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 A␤ and attenuation of A␤ neurotoxic effects.Alzheimer's disease is characterized neuropathologically by amyloid deposits, neurofibrillary tangles, and selective neuronal loss. The major component of the amyloid deposits is amyloid-␤ (A␤), a 39 -43 residue peptide. Soluble forms of A␤ generated from cleavage of amyloid precursor protein are normal products of metabolism (1, 2). The importance of residues 1-42 (A␤42) in Alzheimer's disease was highlighted in the discovery that mutations in codon 717 of the amyloid precursor protein gene, presenilin 1 and presenilin 2 genes result in an increased production of A␤42 over A␤1-40 (A␤40; Refs. 3-5). These results in conjunction with the presence of A␤42 in both mature plaques and diffuse amyloid (6) lead to the hypothesis that this more amyloidogenic species may be the critical element in plaque formation. This hypothesis was supported by the fact that A␤42 deposition precedes that of A␤40 in Down's syndrome (7, 8), in PS1 mutations (9) and in hereditary cerebral hemorrhage with amyloidosis (10).Many in vitro studies have demonstrated that A␤ can be neurotoxic or enhance the susceptibility of neurons to excitotoxic, metabolic, or oxidative insults (11-14). Initially it was thought that only the fibrillar form of A␤ was toxic to neurons (15-18) but more thorough characterization of A␤ structures demonstrated that dimers and small aggregates of A␤ are also neurotoxic (19,20). These data suggested that prevention of A␤ oligomerization would be a likely strategy to prevent AD-related neurodegeneration. Several studies have demonstrated that in vitro A␤-induced neurotoxicity can be ablated by compounds that can increase neuronal resistance by targeting cellular pathways inv...

Among its varied functions, Notch signaling is involved in peripheral T cells responses. The activation and polarization of CD4+ T cells toward a Th1 lineage are essential steps in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Inhibition of all four Notch receptors with a γ-secretase inhibitor was shown to block Th1-type polarization and to attenuate the symptoms of experimental autoimmune encephalomyelitis. In this study, we have examined the role of individual Notch receptors in proliferation, cytokine production, and encephalitogenic potential of PLP-reactive T cells. Specific induction of Notch1 and Notch3 transcripts were noted in PLP-reactive T cells upon Ag stimulation. However, using γ-secretase inhibitor and Abs blocking distinct Notch receptors, we have found that selective inhibition of Notch3, but not Notch1, receptor abrogated proliferation, Th1- and Th17-type responses of PLP-reactive T cells. Moreover, Notch3 inhibition in T cells correlated with the down-regulated expression of protein kinase Cθ, a kinase with important regulatory function within mature T cells. Thus, selective inhibition of the Notch3 receptor may have important effects on peripheral T cell responses and may offer a new attractive target in treating autoimmune diseases, including multiple sclerosis.

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.

We demonstrate for the first time the immunoregulatory potential of transdermal immunization with myelin peptides in MS patients.