| In neurodegenerative diseases in general and in amyotrophic lateral sclerosis (ALS) in particular, discoveries of disease-causing changes to genes have not often translated into an understanding of changes in protein function. For example, the nature of the "toxic gain of function" causing a familial form of ALS (fALS) that results from over one hundred mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) is still a matter of debate.;Whether increased protein aggregation represents this gain of function in ALS, or in any neurodegenerative disease, is unknown. Recent studies have developed algorithms to predict how a given mutation affects protein aggregation propensity. I applied an aggregation rate predicting algorithm to fALS-causing SOD1 mutations and found that aggregation rate is related to ALS patient survival. In a second finding, loss of protein stability (increased likelihood that a protein will unfold) is shown to be related to ALS patient survival. Thus I demonstrate herein that two synergistic properties, namely, increased protein aggregation propensity and decreased protein stability, are central to ALS etiology, and taken together account for 69% of the variability in mutant SOD1-linked fALS patient survival times (Chapter 2). These results are used to rationalize aspects of neurodegenerative disease pathogenesis, including the selective vulnerability of particular neurons and late disease onset. For example, aggregation is a concentration-dependent process, and spinal cord motor neurons have higher concentrations of SOD1 than the surrounding cells. Protein aggregation, therefore, is expected to contribute to the selective vulnerability of motor neurons in fALS.;Next, I investigated the effects of Cu and Zn binding on SOD1 stability using top-down mass spectrometric hydrogen/deuterium exchange (HDX), which allows the simultaneous HDX analysis of multiple SOD1 isoforms [e.g with (holo) and without metal ions (apo)]. The bindings of metal ions were found to significantly affect SOD1 structure, and to an even greater extent than mutation per se. Calculations of the residual deuterium incorporation revealed extensive global disturbance of the apo SOD1 structure rather than limited disorder in metal-binding regions (Chapter 3). |
