The long-term objective of our research is to elucidate pathogenic mechanisms of neuropsychiatric systemic lupus erythematosus by studying neuroimmunologic disease in autoimmune MIRL-Ipr mice. Lymphoid cell infiltration into the choroid plexus, neuronal atrophy, CSF neurotoxicity and an anxiety/depessive behavioral state in MRL-Ipr mice suggest that cytotoxic cells and metabolites in the CSF accelerate apoptosis in limbic regions, thus accounting for altered performance in tasks reflective of emotional reactivity and motivation. The present project aims to examine: 1. Whether DNA fragmentation involves neurons, glial and/or endothelial cells (will be achieved by combining immunofluorescence with TUNEL staining). 2. Whether population of periventricular brain stem cells is susceptible to neurotoxic effects of CSIF (will be achieved by culturing neurospheres and assessing the effects of incubation with CSF from MRL-Ipr mice). 3. Whether brain cell death involves apoptotic pathways (will be achieved by examining nuclear morphology with electron microscopy, by detecting DNA laddering with chemilumnescent method and caspase activation with immunohistochemistry). 4.whether immunosuppression prevents neurodegeneration and CSF neurotoxocity (will be achieved by prolonged treatment with cyclophosphamide, planimetric measurement of affected brain areas, Fluoro Jade B staining and incubation of C1 7.2 stem cells with CSF). 5. Whether the CSF from MRL-Ipr mice contains a distinct proteomic profile (by using profiling proteomic approach with microsystem electrophoresis / nanoelectrospray mass spectrometry, followed by generation of differential protein expression maps). The functional importance of immune and neuropathologic changes will be assessed in all experiments by concurrent measurement of behavioral performance in a well-established behavioral battery. This project, geared towards identification of subsets of potentially neurotoxic cells and mediators, is considered developmental because the P.I. is at an initial stage of introducing molecular and imaging techniques as integral parts of his work. In combination with future direct approaches (aimed to prove causeeffect relationship) it may reveal important pathogenic cascade(s) and represent a qualitative leap in our understanding of autoimmunity-induced neurodegeneration and behavioral dysfunction.
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