Familial amyotrophic lateral sclerosis (FALS) patients possessing any one of 35 known mutations to Cu,Zn superoxide dismutase (SOD) stand a >85% chance of developing this fatal neurodegenerative disease. These SOD mutants invariably cause motor neuron disease when expressed in transgenic mice despite the coexistence of the normal complement of wild-type SOD. The dominant effect of these mutants suggests a gained deleterious function rather than simple loss of superoxide dismutase activity. Evidence from this laboratory indicates that SOD mutants have decreased affinity for zinc in a rank order which appears to correlate with virulence. These observations are combined with the demonstration that zinc-deficient SOD catalyzes protein tyrosine nitration by peroxynitrite more efficiently than Zn-containing SOD. Furthermore, neurofilament-L, a protein critical to motor neuron survival, is a major target of SOD catalyzed nitration and binds zinc but not copper. Lastly, peroxynitrite treatment assembly of triplet neurofilament proteins in vitro, presumably by introducing a permanent negative charge on hydrophobic tyrosine. Thus, it is hypothesized that the selective motor neuron death characteristic of FALS results from the aberrant metal binding properties of SOD mutants which directly or indirectly lead to nitration of neurofilaments and subsequent inhibition of function.The following specific aims are proposed to characterize further metal binding-binding by SOD mutants under physiological relevant conditions, explore mechanisms by which metals may be lost from mutants in vivo, and elucidate mechanisms of peroxynitrite-induced neurofilament dysfunction.
Specific Aim 1) Assess zinc and copper binding by SOD mutants, wild type, and zinc deficient forms under conditions of turnover and exposure to nitric oxide and peroxynitrite.
Specific aim 2) Measure the zinc and copper binding properties of relevant proteins like neurofilaments and metallothioneins which bind metals in vitro and determine whether these proteins can alter the metal content of SODs by direct interaction or by acting as metal sinks.
Specific aim 3) Determine the relative contribution of nitration of individual neurofilament proteins to inhibition of triplet neurofilament assembly in vitro and the extent of nitration required for inhibition.
