Glutamate-provoked excitotoxicity may contribute to the pathogenesis of both acute and degenerative neurological disease. Although glutamate re-uptake is the normal manner through which the action of the neurotransmitter is terminated, glutamate can also be eliminated through enzymatic degradation. We have recently created a functionally active hybrid protein that links a glutamate-degrading enzyme (glutamate-pyruvate-transaminase, GPT-also known as ALT) with the non-toxic neuronal binding domain of tetanus toxin (tetanus toxin fragment C or TTC). The rationale for producing this molecule is to deliver an enhanced capacity to enzymatically-degrade glutamate to synaptic regions surrounding motor neurons, and thereby protect them from toxic levels of glutamate. Our previous studies have demonstrated: 1) TTC can dramatically enhance (100 to 1,000 fold) the delivery of active enzyme to neurons in vitro and motor neurons in vivo. 2) TTC-linked enzymes are targeted to synaptic regions surrounding neurons in vitro and motor neurons in vivo. 3) GPT is the most effective enzyme to rapidly (within minutes) reduce neurotoxic levels of glutamate. 4) GPT can protect neurons in vitro from both direct exposure to toxic levels of glutamate and the toxic effect of inhibition of glutamate re-uptake. We propose to assess TTC-GPT in models of chronic glutamate excitotoxicity and motor neuron degeneration. Initially, we will quantitate the uptake and persistence of active enzymes to explant cultures of neo-natal rat spinal cord with both native GPT and TTC-GPT. We will then assess the capacity of TTC-GPT to prevent progressive motor neuronal death in these spinal cord cultures exposed to inhibitors of glutamate re-uptake, or after reduction of synthesis of high-affinity glutamate transporters using antisense oligonucleotides. We will assess the capacity of TTC-GPT to deliver active enzymes to motor neurons from an intramuscular injection, via retrograde axonal transport, in both normal mice and in a murine model of familial ALS. This proposal will determine the potential of not only glutamate degradation as an anti-excitotoxic strategy, but of TTC as a vector to deliver therapeutic proteins with synaptic sites of action. If successful, TTC-GPT can be assessed in animal models of motor neuron disease, such as the FALS mouse, in the future. These studies will advance understanding of the potential of a novel means of glutamate elimination with therapeutic implications of diseases for not only ALS, but also other neurologic diseases such as stroke, Alzheimer's Disease and Parkinson's Disease.
| Francis, Jonathan W; Figueiredo, Dayse; vanderSpek, Johanna C et al. (2004) A survival motor neuron:tetanus toxin fragment C fusion protein for the targeted delivery of SMN protein to neurons. Brain Res 995:84-96 |
| Matthews, C C; Zielke, H R; Parks, D A et al. (2003) Glutamate-pyruvate transaminase protects against glutamate toxicity in hippocampal slices. Brain Res 978:59-64 |
