A55 CELL-MEDIATED DELIVERY OF GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR (GDNF) IS NEUROPROTECTIVE FOR MYENTERIC NEURONS

Transmural inflammation of the intestine causes structural and functional damage to the enteric nervous system (ENS), that contributes to dysmotility. Elsewhere, neurotrophins can promote neuronal survival and recovery from damage, but a similar role for GDNF, the principal neurotrophin for the ENS, is poorly understood. We investigated the influence of cell-mediated GDNF delivery on myenteric neuronal survival and axonal integrity in the presence of selective neurotoxic challenges which mimic key aspects of inflammation. Enhanced survival due to GDNF was correlated with suppression of intracellular mechanisms of neuronal cell death. A co-culture model of myenteric neurons, smooth muscle and glia from rat neonatal intestine was used to explore effects of structural damage or ischemia on neuronal integrity using immunocytochemistry to visualize myenteric neurons and axons. GDNF was added exogenously or via human embryonic kidney 293 (HEK) cells transfected with a rGDNF-I-hGFP adeno-associated virus plasmid. Mechanisms of cell death were studied using Z-VAD (50 µM) or NEC-1 (100 µM), inhibitors of apoptotic or necroptotic neuronal cell death, and verified by western blotting for key intracellular targets. Mechanical axotomy upon isolation typically caused a 53±3% loss of enteric neurons by 48 hr. The addition of GDNF-GFP Tx-HEK cells prevented this entirely (p<0.05, n=4), and also increased axonal density by 2.8-fold (p<0.05, n=4), while addition of untransfected, control HEK-293 cells had no effects on neuronal parameters. The nitric oxide donors SNP (200 µM) and DETA-NONO (300 µM) caused selective loss of 43±8% and 57±5% of neurons respectively and this was prevented by either NEC-1 (inhibitor of RIP-1 kinase) or by GDNF-GFP Tx-HEK cells. (p<0.05, n=3–4). SNP and DETA-NONO reduced axon density by 38±3% and 44±2% but GDNF-GFP Tx-HEK cells (p<0.05, n=3–4) prevented the decrease. Metabolic inhibition by 2,4-dinitrophenol (DNP; 0.75 mM) reduced neuron number by 55±7%, but was abrogated via GDNF-GFP Tx-HEK cell addition (p<0.05, n=4). Overall, the effects of GDNF-GFP Tx-HEK cells were similar to exogenous GDNF (50 ng/mL), a maximally effective concentration as shown earlier. A cell-mediated source of GDNF is highly effective in preventing neuronal loss or damage in vitro. This suggests that a similar approach in vivo that uses induced expression of GDNF may support the ENS in diverse disease conditions. Inhibitors of apoptosis and necroptosis mitigate neuron loss from axonal damage Robust axonal outgrowth in an established neonatal myenteric co-culture NSERC

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