Effects of c‐Jun N‐Terminal Kinase(JNK) on Neurite Growth

Objective JNK (c‐Jun N‐terminal Kinase) is a member of MAPK (mitogen‐activated protein kinase) family which phosphorylates various transcription factors in nucleus and also cytoplasmic targets. We examined the contribution of JNK signaling to SGN neurite growth by transfection with nutant dominant negative JNK (dnJNK) plasmids in vitro. Method Dissociated spiral ganglion cultures were prepared from P4‐5 rat pups, plated on polyornithine‐ and laminin‐coated 8‐well culture chambers, and maintained in high glucose Dulbecco’s Modified Eagles’s Medium with N2 supplement in a humidified incubator with 6.5% CO 2 . After 1 hour, BDNF and NT‐3 were added to support SGN survival during transfection. Two hours later, the cultures were transfected with expression plasmids, dnJNK1 or dn JNK2 or combination of dn JNK1 and 2, using calcium‐phophate precipitation as previously described. As a negative control, we used pc DNA3 and as a positive control, we used MKK7‐JNK1 plasmid which is a potent neurite growth enhancer. GFP (green fluorescene protein) was stimultaneously added to all culture chambers to be an indicator of transfected neuron. Results Transfection of the constitutively active MKK‐JNK‐1 isoform apparently promotes SGN neurite growth, and co‐transfection significantly reduced SGN neurite growth, whereas transfection of either dnJNK1 or JNK2 singly failed to reduce neurite growth. Thus, JNK1 and JNK2 may serve redundant functions in stimulating SGN neurite growth. SP600125, a small molecule kinase inhibitor, strongly inhibits SGN neurite growth.