The Isolectin IB4 binds RET Receptor Tyrosine Kinase in microglia

Ret receptor tyrosine kinase is the signaling component of the receptor complex for the family ligands of the glial cell line-derived neurotrophic factor (GDNF). Ret is involved in the development of enteric nervous system, of sympathetic, parasympathetic, motor and sensory neurons and it is necessary for the postnatal maintenance of dopaminergic neurons. Ret expression has been as well demonstrated on microglia and several evidence indicate that GDNF regulates not only neuronal survival and maturation but also certain functions of microglia in the brain. Here we demonstrated that plant lectin Griffonia (Bandeiraea) simplicifolia lectin I, isolectin B4 (IB4), commonly used as a microglial marker in the brain, binds to the glycosylated extracellular domain of Ret on the surface of living NIH3T3 fibroblasts cells stably transfected with Ret as well as in adult rat brain as revealed by immunoblotting. Further, confocal immunofluorescence analysis demonstrated a clear overlap in staining between pRet and IB4 in primary microglia cultures as well as in adult rat sections obtained from control or postischemic brain after permanent middle artery occlusion (pMCAO). Interestingly, IB4 staining identified activated or amoeboid Ret-expressing microglia under ischemic conditions. Collectively, our data indicate Ret receptor as one of the IB4-reactive glycoconjugate accounting for the IB4 stain in microglia under physiological and ischemic conditions.