Although such assays have not been demonstrated in the fly CNS, assays of motoneuron targeting in the PNS have been previously described (Jarecki and Keshishian 1995). Using these assays
we identified a significant increase in the frequency of ectopic motoneurons within SOD2bwd/Df7145 mutants, consistent with an axonal targeting defect. Importantly, this ectopic outgrowth phenotype Inhibitors,research,lifescience,medical is rescued with the transgenic SOD2 construct (Fig. 7). In agreement with our data demonstrating that a modest increase in mitochondrial ROS in SOD2bwd/+ heterozygotes (Fig. 5), we see a modest but significant increase in the frequency of ectopic neuronal targeting in SOD2 selleck heterozygous animals as well (Fig. 7). Figure 7 Increase in ectopic targeting of motoneurons in SOD2bwd mutants. (a) Diagram describing
layout of bodywall muscles and motoneurons in the third instar larvae. Muscles are labeled by number. TN is the transverse nerve. The yellow box identifies muscles … Discussion SOD2 proteins perform a critical function in antioxidant defense within mitochondria. Inhibitors,research,lifescience,medical This function is required for health and viability with age, especially within the nervous system, and loss of this function is known to be deleterious. Using a forward genetic Inhibitors,research,lifescience,medical approach we positionally cloned and identified a novel pathogenic mutation affecting SOD2 in Drosophila. This novel mutant, SOD2bwd, exhibits phenotypes known to be associated with SOD2 dysfunction, including reduced longevity and neurodegeneration. However, our studies of SOD2bwd demonstrate that this mutant results
in reduced steady Inhibitors,research,lifescience,medical state protein levels, stress-sensitive paralysis, neurodevelopmental defects, neuropathology, and aberrant axonal targeting not previously associated with SOD2 dysfunction. Surprisingly, modeling of the missense Inhibitors,research,lifescience,medical mutant does not predict an alteration in the structure of the SOD2 protein despite an amino acid substitution (G138D) that is different in both size and charge. These structural studies employed multiple protein modeling algorithms, which failed to produce MycoClean Mycoplasma Removal Kit a structure with significant alterations. These findings are from in silico studies and it remains possible that a structure derived by other methods, such as from a protein crystal, may identify changes that were not observed by these methods. Nonetheless, these studies suggested that the mutant protein could retain wildtype function and that altered stability of the protein might underlie SOD2bwd pathogenesis. Consistent with this conclusion, steady state SOD2 protein levels were examined by Western blot and revealed a significant reduction from that of controls. These studies suggest that the mutant protein is unstable; however, additional experiments (e.g., pulse chase studies) to directly measure protein stability will be needed to verify that the reduced protein levels are the result of altered protein stability.