We respond to the stated goal of the RFA: NS-01-004, to: "Understand why motor neurons are selectively vulnerable to the toxic effects of certain gene mutations." We showed that the expression of the G93A-mutation of the Cu, Zn superoxide dismutase gene (SOD1 G93A) of familial ALS (FALS), by transfected cells, is followed by the fragmentation of the Golgi apparatus (GA) and cell death. The fragmentation of the GA is identical to that reported in motor neurons in human sporadic ALS (SALS), in FALS with SOD1 mutations, and in transgenic mice expressing SOD1 G93A where fragmentation of the neuronal GA was observed months before the onset of paralysis; in these mice, mutant protein aggregated in bundles of 13 nm thick pathological filaments, the appearance of which correlated with disease progression and fragmentation of the neuronal GA. We hypothesized that the 13 nm-thick filaments contained not only SOD1 G93A, but also "trapped" or "modified" proteins required for the structural organization of the GA. Pursuing this hypothesis, we propose here to develop a method for the isolation of enriched fractions of bundles of pathological filaments from spinal cords of transgenic mice expressing SOD1 G93A, in order to perform protein analyses. The isolation of the filaments will be accomplished in two steps; first by a sucrose density gradient separation, followed by immunoisolation with antibody-coated magnetic beads. Protein and peptide analyses will be performed with standard methods used previously in the laboratory in studies on MG 160, the medial Golgi sialoglycoprotein we discovered. This application aims at the optimization of the method for purity and yielding of filament bundles. The preliminary results are very encouraging and, in the near future, we will apply for the structural and functional characterization of the polypeptides-constituents of the 13 nm-thick filaments. Although different etiologies cause sporadic ALS (sALS) and FALS with SOD1 mutations, identical pathogenetic mechanisms, such as fragmentation of the neuronal GA, may be involved in both. Fragmentation of the neuronal GA occurs early in the chain of events causing neuronal degeneration, and it may be an important event considering that over 45 proteins destined for fast axoplasmic transport are transported, processed and targeted by the organelle.
NIH ExPorter
NIH ExPorter