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GDNF PROMOTES AXONAL PLASTICITY OF THE POSTNATAL ENTERIC NERVOUS SYSTEM (ENS)
DM Rodrigues, SR Lourenssen, MG Blennerhassett
GIDRU, Department of Physiology, Queen’s University, Kingston, Ontario
BACKGROUND: Neurotrophins from target cells allow neurons to survive, develop and remain plastic in the central nervous system, but very little is known about their presence and roles in the postnatal ENS. However, the rapid axon outgrowth seen following neural damage in TNBS-induced colitis in the rat suggests their active role in inflammation. HYPOTHESIS: Since glial cell-derived neurotrophic factor (GDNF) is involved in ENS development, we hypothesized that GDNF also promotes axon growth in myenteric neurons of the postnatal rat intestine, acting through its receptors GFRalpha1 and RET. We also speculated that GDNF expression would increase at the onset of axon growth in the post-TNBS animal, possibly regulated by proinflammatory cytokine TNFalpha.
Methods and RESULTS: Western blotting of the smooth muscle and myenteric plexus (SMMP) from postnatal day 2-4 and adult rats showed the presence of GDNF and its receptors, GFRalpha1 and RET. To assess the role of GDNF, we used a co-culture model of postnatal rat myenteric neurons and smooth muscle. Co-cultures were established in DMEM or DMEM containing 100 ng/mL GDNF, and immunocytochemistry was used to detect neuronal cell bodies and axons. GDNF increased axon density by 64% (p<=0.05) over the control group, suggesting a direct role of GDNF on axon growth. GDNF treatment caused an increase in pRET, indicating that GDNF was signaling through a GFRalpha1/RET dependent system. Exogenous treatment of TNFalpha (50ng/ml) increased GDNF expression in co-cultures, suggesting a role of proinflammatory cytokines in promoting neurotrophin expression during inflammation. However, western blotting determined that GDNF expression was sharply reduced on Days 2-6 of TNBS colitis, but returned to normal by day 35.
CONCLUSIONS: GDNF and GFRalpha1 are present in the rat myenteric plexus, and GDNF enhances axon growth in cultured postnatal SMMP through GFRalpha1/RET. Although GDNF expression was increased by TNFalpha in vitro, GDNF expression was decreased in vivo during colonic inflammation. This suggests that either GDNF may not be responsible for axon growth during colitis in the adult rat, or conversely, the decrease in GDNF may reflect inadequate amounts of neurotrophin that could underlie inflammation-induced axon and neuron damage in the ENS.
Supported by CCFC