The development of new therapies to treat disorders of the central nervous system (CNS) is often hindered by the poor bioavailability of candidate drugs (e.g. small molecules, peptides, proteins, or viruses) to target cells within the brain and/or spinal cord. In particular, recombinant glial cell line derived neurotrophic factor (GDNF) has been shown to have robust neuroprotective and neurorestorative effects in a variety of animal models for neurological disease and CNS injury in vivo, however, its success in treating human neurological disorders has been limited by its poor delivery to the much larger human brain. The overall goal of this project is to evaluate the therapeutic potential of a novel recombinant fusion protein composed of GDNF linked to the neuronal binding fragment of tetanus toxin (tetanus toxin fragment C, or TTC). Our study has four specific aims: (1) design, express, and purify GDNF:TTC fusion proteins; (2) characterize the functional activity of these fusion proteins; (3) determine the distribution, persistence, and tissue levels of GDNF:TTC fusion proteins in the CNS following intraparenchymal, intraventricular, and intramuscular administration; and (4) investigate the neuroprotective activity of the fusion proteins using a mouse model of Parkinson's disease. The experiments in Aim 1 will involve the use of cDNA constructs (all currently in hand) and recombinant protein purification techniques. The studies in Aim 2 will employ primary neuron cell culture in conjunction with immunocytochemical, enzyme immunoassay, and morphometric techniques to assess the functional properties of the GDNF:TTC fusion proteins in vitro. The studies in Aim 3 will similarly use immunocytochemical and enzyme immunoassay methods to investigate the delivery of fusions proteins to mouse and rat CNS in vivo. Finally, the experiments in Aim 4 will use a neurochemical approach to investigate the neuroprotective effects of GDNF:TTC in mice in vivo. While the present work may give rise to a new treatment for Parkinson's Disease, the information obtained from our characterization of GDNF:TTC may lead to its use in other neurological disorders such as amyotrophic lateral sclerosis.(ALS). ? ?
