The enzyme superoxide dismutase (SOD) is under active investigation as a form of neuroprotective therapy in both neurodegenerative diseases and brain injury. Increasing cellular levels of SOD have increased neuronal viability in several models of free radical mediated injury. Poor penetration of SOD into the nervous system limits its utility as a potential therapeutic. The non-toxic binding fragment of tetanus toxin, termed C-fragment (CF), shares many characteristics of its parent protein, such as entry into and persistence within the nervous system. These properties have led investigators to demonstrate its ability to be used as a vector to deliver large proteins including enzymes to the nervous system in experimental animals. Using recombinant genetic methods we have produced a hybrid protein containing CF and SOD. This protein retains the neuronal binding properties of CF and the enzymatic properties of SOD. This proposal will explore the potential of this hybrid protein as a novel neuroprotectant. Using both a cell line and cultured embryonic neurons we will determine to what extent CF-SOD can raise cellular levels of SOD. We will then determine if cells treated with CF-SOD are resistant to damage and death due to exposure to toxic levels of the excitatory amino acid glutamate. In animals we will determine the extent to which CF-SOD enhances the nervous system uptake SOD. Using radiolabeled proteins we will compare uptake of native SOD and CF-SOD after intravenous, intramuscular and intrathecal administration. We will determine the amount of distribution and lifetime of these radiolabeled proteins within the nervous system. The effect of anti-tetanus antibodies on nervous system uptake of CF-SOD will be examined in both previously immunized rats and rats receiving sera from immunized humans. SOD levels will be measured enzymatically within the nervous system after administration of CF-SOD. We will determine to what extent CF-SOD can raise cellular levels of SOD. Using subcellular fractionation techniques, we will determine what cellular compartment accumulates SOD activity of administration of CF-SOD. These experiments will explore the biology of this unique protein in the nervous system and evaluate its potential as a neuro-protectant. Successful completion of this proposal will provide the basis for preclinical nd clinical study of CF-SOD as a new therapeutic agent for neurological disease.
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