Cellular pathology of Alexander Disease

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We propose to examine several aspects of intermediate filament (IF) organization and cellular stress reactions that may be germane to an understanding of the pathophysiology of Alexander disease. Thus, the over-expression of IF proteins may lead to an abnormal organization of filaments, which subsequently leads to up-regulating the transcription and translation of the small hsps, alphabeta-crystallin and hsp27. We must also consider that Alexander disease is a disorder in which a mutant gene expressed on one cell type (astrocyte) results in degeneration and/or abnormal development of another cell type (oligodendrocyte). We propose three specific aims: 1) How are mutant GFAPs expressed in cells and do they result in an abnormal organization of IFs? Is there an accumulation of IFs? Does the expression of mutant GFAPs alter IF turnover. 2. Does the accumulation of IFs lead to a cellular stress response, part of which leads to the up-regulation of small hsps? If so, what are the mechanism(s) that produce such a stress response? If IF accumulation induces small hsps, we will investigate intracellular stress signal pathways that may underlie this effect, focusing on HSF1 activation, MAP kinase activation (ERK1/2, JNK, p38 kinase), NF- kappaB activation, and protein kinase-N activation. 3. How does the expression of a mutant protein in one cell type in the CNS (astrocytes) produce deleterious effects on another cell type (oligodendrocytes)? Is the cellular stress response an important part of this link? Do the pathological changes in astrocytes interfere with oligodendrocyte differentiation and/or myelination? We will examine possible further consequences of IF aggregation, focusing on the possibility that "stressed" astrocytes regulate cytokines. These experiments will focus on how the accumulation of a protein in one cell type (Astrocytes) appears to produce deleterious effects on another cell type (oligodendrocytes), the rationale taken from experiments in which cytokines are toxic to oligodendrocytes. In addition, we will examine other potentially toxic substances, including reactive oxygen species. If the neuropathology of the transgenic mice that is constructing suggest a defect in oligodendrocyte development and/or myelination, then we will examine directly the interactions between astrocytes expressing GFAP mutations and oligodendrocyte progenitors using a cell culture system.